Abstract Background Temporal changes in HIV-1 resistance both reflect and influence the clinical use of antiretrovirals (ARVs). Objective To determine temporal trends in reduced susceptibility to ARVs and resistance mutations in routine clinical samples (RCS) from HIV infected patients. Study design Calculated fold-changes (FC) for ARVs were determined for viral genotypes from RCS received between July 1998 and June 2007 using vircoTYPE HIV-1 (Version 4.2.01). The prevalence of isolates with clinically relevant reduced susceptibility and of resistance mutations were determined for consecutive 6-month periods. Results 242,003 RCS were identified. The prevalence of RCS exhibiting reduced susceptibility to ≥1 drug among any of three ARV classes decreased from 82% to 66.5% and of these, to ≥1 drug in each of the three ARV classes and from 30.5% to 15.7% from December 98 to June 07 ( p ≤ 0.0001). The prevalence of mutations associated with NRTI, NNRTI and PI resistance generally reflected evolving use of these drug classes. Among fully susceptible RCS, isolates that exhibited resistance mutations were rare. Conclusions Clinically relevant reduced susceptibility to ARVs declined over the 9-year period. There was a general higher prevalence of reduced susceptibility to RT inhibitors than to PIs.