Abstract The molecular epidemiology of HIV-1 infection was first studied in Cyprus in the mid-1990s, but the extent of HIV-1 diversity and the prevalence of drug resistance have remained elusive. In an effort to address this issue, the present study examined HIV-1 strains isolated from 37 newly diagnosed untreated HIV-1 patients, representing 72% of the total number of newly diagnosed and drug-naive patients in the period 2003 to 2006. DNA sequences encoding the gag (p17, p24, p2, p7, p1, and p6), pol (protease and reverse transcriptase), and env (gp160) regions were amplified by RT-PCR from plasma HIV-1 RNA from all patients and sequenced using a newly designed methodology. All amplified products were studied according to established genetic methodologies to determine the genetic subtype and the prevalence of drug-resistance-associated mutations to currently available antiretroviral drugs. Analyses of the obtained viral sequences indicated that subtype A was the most common subtype present and accounted for 38% of the infections followed by subtype B (35%), subtype C (13%), CRF02_AG (8%), and subtypes D and CRF01_AE (3% each). One patient (2.7%) had an M41L/M and another patient (2.7%) an M184V amino acid substitution in the reverse transcriptase (RT) associated with high-level resistance to RT inhibitors. There were no patients with resistant mutations to protease inhibitors (PI). Additionally, one patient (2.7%) had an L44M amino acid substitution within the HR1 region of gp41 conferring resistance to the enfuvirtide (T20) fusion inhibitor. Similar to results of the 1994 molecular epidemiological study, these data demonstrate the extensive heterogeneity of HIV-1 infection in Cyprus and the low prevalence of transmitted resistance to current HIV-1 antiretroviral drugs. Taken together, these findings demonstrate that HIV-1 infection in Cyprus is being replenished by a continuous influx of new strains from many countries, establishing an ever-evolving and polyphyletic infection in the island.