The viral hit - recently remixed with Billy Ray Cyrus - has now topped the Billboard Hot 100 for 17 weeks.
Old Town Road has had 72.5 million US streams and 46,000 downloads in the past seven days.
All the different versions of the song are close enough to the original, and so count towards one single total.
The track beat the previous 16-week record, held jointly by the 2017 version of Despacito, by Luis Fonsi, Daddy Yankee and Justin Bieber, and Mariah Carey and Boyz II Men's 1995 track One Sweet Day.
Billie Eilish's song bad guy remains number two in the US charts for the seventh straight week, unable to topple Old Town Road from its place.
Lil Nas X - real name Montero Lamar Hill - was an unknown artist who first released the track on Soundcloud in December 2018.
Boasting a music video with footage from Western video game Red Dead Redemption II, the song went viral on TikTok.
Old Town Road has spent 18 weeks on the UK singles chart, two of them in the top spot.
In March, the song was controversially removed from Billboard's country chart in the US despite reaching the top 20 for allegedly not doing enough to "embrace enough elements of today's country music".
Several commentators suggested race was a factor in Billboard's decision to eliminate Lil Nas X's chart entry.
The artist also recently revealed he has experienced a backlash on social media since posting cryptic hints on Twitter that he is gay.