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The objective of this study was to investigate the incidence, demographics, HIV subtype, and genotypic resistance of acute HIV infections in a high-risk Thai population.Between March 2006 and September 2007, 6426 stored samples at the Thai Red Cross Anonymous Clinic were screened for acute HIV infection by 2 methods: pooled nucleic acid testing (NAT) of fourth-generation enzyme immunoassay (EIA)-negative samples (n = 5402) and subsequent first-generation EIA testing of fourth-generation EIA-positive samples (n = 1024).Eleven acute HIV-infected subjects were identified by pooled NAT (n = 7) and serial EIA (n = 4). Mean age was 28 years; 9 were male; and 60% were men who have sex with men. Median HIV RNA was 99,601 copies per milliliter (log10 viral load (VL) = 5.00). Eight samples could be genotyped: 6, CRF01_AE; 1, subtype B; and 1, CRF01_AE/B recombinant. No resistance to antiretroviral therapy was found. The HIV incidence per 100 person-years, calculated from the pooled, antibody-negative samples, was 2.7% (95% confidence interval, 2.2%-4.3%).This is the first report of antibody-negative, NAT-positive, acute HIV infection in Thailand. The majority were men who have sex with men, which reflects the current epidemic in Thailand and justifies prevention programs aimed at this group. This high-risk population may be suitable for future studies on acute HIV infection, HIV treatment, vaccine, and prevention of onward transmission strategies.