High-resolution HIV-1 genotyping of large sample sets is crucial to define the evolving and dynamic epidemics in Asia. Here we present MHAbce v.2, a multi-region hybridization assay that individually discriminates subtypes B, C, CRF01_AE, and virtually all of their described recombinants, based on real-time PCR using subtype-specific TaqMan probes in 8 regions throughout the viral genome. In a validation panel (n=70), the assay performed with a sensitivity of 95.7% and specificity of 99.8%. The assay was field-tested on samples from a retrospective MTCT cohort (n=180; Lampang Province, Northern Thailand; 1996-1998). 177/180 of the samples were typeable, and 94.4% were typed as CRF01_AE. The remaining strains represented even proportions of subtype B and B/CRF01_AE recombinants and were confirmed by sequencing, revealing early links between the heterosexual and IDU HIV-1 epidemics in Thailand. MHAbce v.2, with an area of application including China, India, Southeast Asia, and the Pacific Rim, can be used to develop a comprehensive and detailed picture of this important component of the HIV/AIDS pandemic.