Guitar Hero has made several appearances in popular culture. Guitar Hero II is the centerpiece of an episode of South Park titled "Guitar Queer-O", in which Stan and Kyle overindulge in Guitar Hero II and become treated as though they were real-life rock stars. The episode was first broadcast on November 7, 2007, ten days after the American release of Guitar Hero III: Legends of Rock. Metalocalypse (created by Titmouse Studio, who also provided cutscene animations within the Guitar Hero game) has made reference to Guitar Hero; in one example, the episode "Dethkids," a sick child composes a song for Dethklok rhythm guitarist Toki Wartooth using a Guitar Hero controller. Ellen DeGeneres has played Guitar Hero several times in 2008 during the monologue of her syndicated talk show. In the January 25, 2008, episode she is seen playing along to "Barracuda", which segues to Heart performing the song for the audience. During the season 7 finale of American Idol, finalists David Cook and David Archuleta appeared in separate commercials for the games, where they each parodied Tom Cruise dancing to "Old Time Rock and Roll" in the movie Risky Business. This parody has been used by director Brett Ratner to create advertisements for Guitar Hero World Tour, featuring celebrities such as Kobe Bryant, Tony Hawk, Alex Rodriguez, Michael Phelps, Corbin Bleu, Heidi Klum, and Marisa Miller.

Political cartoonist Steve Breen illustrated a cartoon where Queen Elizabeth II received Guitar Hero as a gift from visiting President Barack Obama, a parody of Obama's visit to England in April 2009 during which he gave Queen Elizabeth II an iPod.

A 2009 Close to Home comic strip portrayed a young boy blowing into a tuba-shaped joystick while playing a video game, as his parents cover their ears, with the caption: "Despite the success of Guitar Hero, Tuba Hero bombs big-time."

During Halftime of MLS Cup 2009 two league players, Jimmy Conrad of the Kansas City Wizards and Freddy Montero of Seattle Sounders FC faced off to Michael Jackson's "Beat It" in Guitar Hero World Tour.[83]

A scene, written into the script by Vince Vaughn, in Couples Retreat features Vaughn's character challenging a resort employee to a Guitar Hero 5-faceoff.

MC Lars' This Gigantic Robot Kills includes a song called "Guitar Hero Hero (Beating Guitar Hero Does Not Make You Slash)", poking fun at frequent players of GH. It features a solo from Paul Gilbert. (The song's lyrics reference the following real-guitar players in addition to those two: Robert Johnson, Dimebag Darrell, Eric Clapton and Jimi Hendrix.) Mariah Carey's music video for her song "Touch My Body" features a nerd strumming on a Guitar Hero controller. The music video for San Diego rock band Switchfoot's song "Awakening" featured two men going home and competing separately on Guitar Hero, using stop-motion cardboard cutouts to simulate the video game interface.The music video for Eminem's "We Made You" includes portions of the song where Eminem is singing in front of a Guitar Hero-like note highway. The video for Brad Paisley's video for "Start a Band", featuring Keith Urban, includes a Guitar Hero-like "Guitar Showdown" video game as part of the video's story.

The series' popularity spread to the sports world; Detroit Tigers' pitcher Joel Zumaya injured himself during the 2006 playoffs playing Guitar Hero. In professional wrestling, former TNA Wrestling tag team of Jimmy Rave and Lance Rock, known as The Rock 'n Rave Infection, have a gimmick of dressing as characters from the game and strumming the guitar controllers on their way to the ring.

The name "Guitar Hero" is often juxtapositioned with other instruments or other tools for humorous effects. There are T-shirts available from at least one vendor, parodying the Guitar Hero logo with a number of different instruments, from violin to bagpipes. Marketing for The Simpsons Game included mock posters for "Sitar Hero", depicting The Simpsons character Apu with a controller with 21 buttons, a parody Harmonix praised. GameSpot released an April Fools' article in 2006 announcing "Cowbell Hero" as a successor to the first game, referencing the "More cowbell" skit from Saturday Night Live. Also, a "Cowbell Hero" T-shirt appears among Chuck's collection of shirts in an episode of the NBC series, Chuck. One Penny Arcade comic strip envisioned a game entitled "Photoshop Hero", which has become a design for one of their t-shirts.[94] A 2008 FoxTrot Sunday comic showed the characters Jason and Marcus playing a video game called "Chamber Music Hero."[95] The satirical newspaper The Onion published an article entitled "Activision Reports Sluggish Sales For Sousaphone Hero."

On March 2010, the Office For National Statistics (UK) announced that Guitar Hero will be used to gauge the country's inflation risk. Viacom Inc.'s Rock Band was also included to reflect changes in (UK) consumer spending.

A Guitar Hero reality show could be developed in the future, along with a possible concert tour.

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