A molecular epidemiological investigation was conducted among various risk populations (n = 184) in Kuala Lumpur, Malaysia, in 2003 to 2005, on the basis of nucleotide sequences of protease and reverse transcriptase regions. In addition to circulating HIV-1 strains, including CRF01_AE (57.1%), subtype B (20.1%), and subtype C (0.5%), we detected a candidate with a new circulating recombinant form (CRF). We determined four near-full-length nucleotide sequences with identical subtype structure from epidemiologically unlinked individuals of different risk and ethnic groups. In this chimera, two short subtype B segments were inserted into the gag-RT region in a backbone of CRF01_AE. The recombinant structure was distinct from previously identified CRF15_01B in Thailand. In agreement with the current HIV nomenclature system, this constitutes a novel CRF (CRF33_01B). The overall prevalence of CRF33_01B is 19.0% (35/184). Although the prevalence of CRF33_01B is particularly high among injecting drug users (42.0%, 21/50), it is also detected in a substantial proportion of homo-/bisexual males (18.8%, 3/16) and heterosexuals (9.8%, 9/92). Moreover, unique recombinant forms composed of CRF01_AE and subtype B that have a significant structural relationship with CRF33_01B were detected in 1.6% (3/184) of study subjects, suggesting an ongoing recombination process in Malaysia. This new CRF seems to be bridging viral transmission between different risk populations in this country.