The purpose of this study is to evaluate whether the choice of a PI- or an efavirenz (EFV)-based HAART initial regimen impacts on the viral diversity after failure from a second, class-switch salvage regimen. Sequential HAART failures after a class switch were identified for which the genotypes showed evidence of signature mutations at each failure. Each second failure was required to be from a viral burden <400 RNA c/ml. Thirteen cases of sequential failure from an initial EFV-containing to a PI-containing regimen (EP), and 19 sequential failures from an initial PI-containing to an EFV-containing regimen (PE) were identified. The persistence of signature mutations from the first failure were evaluated at second failure and compared between the EP and PE groups. Phylogenetic trees were constructed for a subgroup of cases from existing genetic sequence information and branch length analysis was used to determine evidence of viral diversity between groups. For EP sequential therapy, 10 of 12 cases carried forward a key non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation in the second failure compared to 5 of 13 cases for PE sequential therapy (p = 0.041). Phylogenetic analysis demonstrated that there was more viral diversity in the PE group as compared to the EP group, consistent with the interpretation that mutations at the second failure added to an ancestral virus closer to baseline rather than to the dominant virus at first failure. The development of HIV viral diversity after multiple HAART failures is determined by the sequence in which the regimens are ordered.