The early detection of human immunodeficiency virus type 1 (HIV-1) infection in infants is complicated by the persistence of maternal antibodies and by diverse HIV-1 subtypes. We developed a nested, three-monoplex HIV-1 DNA PCR (N3M-PCR) assay to detect diverse HIV-1 subtypes in infants born to infected mothers. We optimized the test for use with dried blood spot (DBS) samples for ease of storage and transport from rural China to central laboratories. Six pairs of primers were designed that targeted env, gag, and pol genes, and the test was run in three reactions with an analytical sensitivity of 10 copies DNA per reaction to cover nine HIV-1 subtypes, A, B, C, D, F, G, CRF01-AE, CRF08-BC, and CRF07-BC. The assay performance was evaluated on 347 DBS specimens from 151 exposed infants in four diverse provinces of China in which multiple subtypes were circulating. The results of this test were compared to those of HIV antibody enzyme immunoassay and Western blotting confirmation for the infants at > or =18 months of age or to convincing clinical and epidemiologic data for deceased infants. The sensitivity of the N3M-PCR assay was 30.0% (3/10) for infants at 48 h after birth, 91.7% (11/12) at 1 to 2 months of age, and 93.7% (15/16) at 3 to 6 months of age. The specificity was 100% (94/94) at all three time points. The PCR reproducibility in the three DNA regions was 100% for samples at 48 h after birth, 96.7% at 1 to 2 months, and 100% at 3 to 6 months of age. The HIV-1 DNA N3M-PCR assay on DBSs offers a simple and affordable approach for early infant HIV-1 diagnosis in regions with diverse HIV-1 circulating subtypes.