The I50V protease inhibitor (PI) resistance mutation was found in 87.4% of protease gene fragments sequenced from 199 nucleic acid isolates extracted using an NASBA virus load assay, performed between 1997 and 2001 in Brazil. This mutation is an amprenavir-related mutation, and at that particular time this PI was seldom used in Brazil. This mutation was found both in patients with and without therapeutic success. Q calibrators showed the PI resistance mutation I50V when directly amplified and sequenced from the 423-bp PCR product targeting protease gene. The majority of the patients’ samples had a mixture of I50I and I50V; however, this artifact was nor seen when a 989-bp PCR product was used. These results show that RNA extracted using virus load kits need to be critically evaluated before being used in home-brew genotypic tests.