We identified an HIV-1 isolate with a 3 base pairs insertion in the 100-105 region of the reverse transcriptase gene (RT) along with a G190E and a V75A mutation. Virus carrying the insertion alone or in association with G190A was not infectious. The association of G190E and the 100-105 insertion displayed a high level of resistance to non-nucleoside reverse transcriptase inhibitors; the addition of the insertion to G190E may increase the activity of RT.