The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance mutation K65R confers intermediate levels of resistance to several RT inhibitors, including a three- to fourfold reduction of tenofovir susceptibility. Here, we have used for the first time primary HIV-1 isolates from individuals who developed the K65R mutation while enrolled in a clinical trial of tenofovir to analyze the impact of this mutation on HIV-1 replicative fitness. A marked impairment in replicative fitness was observed in association with the selection of viruses carrying the K65R mutation in all patients. The mean replicative fitness among these viruses was 20% relative to the corresponding baseline wild-type virus, ranging from 10 to 32% depending on the accompanying RT mutations. These results support a reduction in in vivo replication for K65R mutant viruses.