Abstract This study among antiretroviral-experienced prisoners from central western Brazil investigated mutations associated with secondary resistance to nucleoside/nonnucleoside reverse transcriptase inhibitors (NRTI/NNRTI), protease inhibitors (Stanford HIV-1 Resistance/International Aids Society Databases), and HIV-1 subtypes (REGA/phylogenetic analyses/SimPlot). Twenty-seven prisoners from three prisons (16 males and four females from Mato Grosso do Sul State and seven males from Goiás State) had HIV-1 protease and reverse transcriptase fragments sequenced after nested PCR. Median age was 35 years. Seven males and two females were intravenous drug users, three males referred homosexual practice. Resistance mutations were present in 37% (10/27): NRTI+NNRTI mutations (n=5), NRTI mutations (n=3), multidrug-resistant mutations (n=2). Subtype B (48%), subtype C (11%), B/F1, B/C, and F1/B/C recombinants (40.7%) were detected. Possible intraprison transmissions were identified: two intravenous drug user females (subtype C); two clusters among homosexual males (subtype B and B/F1). High resistance rate and possible intraprison transmission highlight the need for improved prevention, counseling, and treatment strategies for prisoners.