The Beatles are among three acts to have two entries in the list's top 25. As well as Yesterday at number 1, their 1964 hit I Want To Hold Your Hand is at number 6.
The ballad, written by Sir Paul McCartney and released in 1965 on the album Help!, edged the Rolling Stones' I Can't Get Number Satisfaction - also released in 1965 - into second place.
Nirvana's 1991 single Smells Like Teen Spirit - an anthem for the grunge era and a requiem for its leading light Kurt Cobain - was third.
The teen pop contingent is also represented, with Backstreet Boys at number 10, with I Want It That Way, Britney Spears at number 25 with Baby One More Time and NSync at number 55 with Bye Bye Bye.
The experts at Rolling Stone and MTV admit they arbitrarily started their list with the emergence of the Beatles in 1963.
They admitted their list was based on songs that had the most influence on popular culture, not necessarily the best songs.
This explains, they say, why Eric Clapton's Layla was not included but his ballad, Tears in Heaven was (at number 45).
Tom Calderone, MTV's senior vice president for music programming said the choice of Yesterday was "an automatic".
An ode to lost love and wistful longing, McCartney's song is familiar to almost every generation.
It has been recorded more than 3,000 times by numerous other artists, from Ray Charles and Frank Sinatra to Boyz II Men.
The Beatles are among three acts to have two entries in the list's top 25. As well as Yesterday at number 1, their 1964 hit I Want To Hold Your Hand is at number 6.
Also with two songs each in the top 25 are the Rolling Stones with (I Can't Get Number ) Satisfaction at number 2 and Brown Sugar at number 14.
Madonna makes it to number 4 with Like a Virgin and to number 19 with Vogue.
Michael Jackson has three entries in the top 25. As a solo artist he charts at number 5 with Billie Jean and at number 22 with Beat It, while the Jackson 5 hit I Want You Back earns him a place at number 9.
In all, there are 16 songs from the 1960s, 27 from the 1970s and 28 each from the 1980s and 1990s. `N Sync's song was from this year.
Rolling Stone is devoting a special issue to the list, and MTV is counting it down in a five-part series starting 20 November.