ABSTRACT: Individuals infected with HIV-1 non-B subtypes are understudied in the United States. Their characterization may augment prevention and treatment interventions. We examined the regional molecular epidemiology of non-B subtypes using a combined phylogenetic and geospatial approach. HIV-1 pol sequences and clinical data obtained for routine clinical care were aggregated from 2004 to 2011 at the largest HIV center in Rhode Island. Subtyping was performed by neighbor-joining and maximum-likelihood phylogeny and compared across eight commonly used tools (HIVdb, REGA, RIP, NCBI, Geno2Pheno, EuResist, jpHMM and STAR) using proportional odds ordinal regression. Individuals with non-B subtypes were characterized according to demographics and risk factors for infection, intra-subtype clustering by maximum-likelihood phylogeny, and geospatial hotspot analysis using Getis-Ord Gi(?) statistics. Of 1277 unique sequences, phylogenetic subtyping demonstrated 8.3% (N=106, 95% CI 6.8-10%) non-B subtypes and circulating recombinant forms (CRFs): CRF02_AG=46; A=15; C=15; CRF01_AE=6; CRF06_CPX=5; CRF14_BG=5; G=3; CRF43_02G=3; D=3; CRF24_BG=3; CRF11_CPX=1; F1=1. Compared to phylogeny, Geno2Pheno was the most concordant (86% exact match) followed by REGA (85%), EuResist (85%) and STAR (82%). Of 106 individuals with non-B subtypes, 50% were male, 71% acquired infection through heterosexual transmission; 76%, were born in Africa, 6% Southeast Asia, 5% the United States, 3% Central America, 1% Europe, and 9% unknown. Eighty percent of CRF02_AG, 93% of A and 87% of C sequences were from African-born individuals. Twenty-two percent of non-B subtypes formed transmission clusters, including a significant number of younger individuals with perinatally-acquired infection. Geospatial analyses revealed hotspots of B and non-B subtypes in the state capital with a more concentrated focus among non-B subtypes. Molecular examination of regional HIV diversity revealed a larger than expected non-subtype B infected population, mostly born in Africa, with low ongoing regional transmission. Phylogenetic and geospatial characterization of infection clusters is helpful to identify targets for treatment and prevention interventions.