BACKGROUND : Guangdong Province is one of the most developed and populous provinces in southern China, with frequent foreign exchanges and large transient population. The annual number of cases of HIV/AIDS reported in Guangdong has been higher than most of provinces in China for several successive years. HIV infection by heterosexual transmission occurs across the province, with transmission among men who have sex with men occurring mainly in larger urban centers. There is a lack of widespread and representative data on the distribution of HIV subtypes in Guangdong. This study aimed to thoroughly investigate and estimate the prevalence and distribution of HIV-1 subtypes using a city-based sampling strategy to better understand the characteristics of HIV transmission in Guangdong. METHODS : Archived plasma samples (n = 1205) from individuals diagnosed as HIV-1 infection in 2013 were selected randomly from all 21 cities in Guangdong Province. Genotypes were determined using env and/or gag sequences using phylogenetic analysis. The distributions of HIV genotypes in different risk groups and different cities were analyzed. RESULTS : A total of 15 genotypes, including six discordant genotypes, were identified. The four main HIV-1 subtypes in Guangdong were CRF01_AE (43.2%), CRF07_BC (26.3%), CRF55_01B (8.5%), and CRF08_BC (8.4%). CRF01_AE was the predominant subtype in all risk populations. The high mobility of people shaped the complexity of the HIV genotypes, while the switch of risk factors affected the distribution and future trend of HIV-1 genotypes in Guangdong. Another epicenter located in the western region in addition to the known epicenter cities in the Pearl River Delta region of Guangdong may exist. CONCLUSIONS : Our study provides a comprehensive molecular epidemiologic dataset to understand the diversity and distribution of HIV genotypes in Guangdong, as well as to clarify the unique region- and risk group-specific transmission dynamics. The results provide critical and insightful information for more effective intervention strategies to limit HIV transmission in the future.