Abstract Honghe Prefecture, Yunnan Province, China, is a key area affected by HIV/AIDS. Given the high number of infected individuals, understanding HIV transmission in this region is crucial to controlling the epidemic in Yunnan Province. A molecular epidemiology study with 1004 newly diagnosed HIV-1 cases in 2022 was conducted to characterize transmission and guide targeted interventions. Of the 833 samples genotyped, nine HIV-1 genotypes were identified, with CRF08_BC (56.7%), URFs (17.8%), CRF07_BC (13.9%) and CRF01_AE (7.1%) being the most prevalent. These genotypes were widely distributed but concentrated in the center and east of the prefecture. Molecular network analysis showed varying clustering rates among counties, with Luxi County having the highest and Honghe County the lowest. Low education levels were associated with higher clustering. Cross-county transmission patterns were revealed, with strong links between specific counties. Of the three spatial subdivisions identified by the cohesive subgroup analysis, subgroup I included eight counties running from northeast to southwest. Persons aged 50–59 years and ethnic minorities were more likely to be identified in cross-county transmission. Notably, the overall prevalent level of HIV-1 pre-treatment drug resistance was 6.2% (50/809), with higher levels in Luxi (18.2%, 8/44), Hekou (14.3%, 3/21) and Yuanyang (10.9%, 13/119). This study investigated HIV-1 transmission patterns using molecular network analysis in a hotspot of HIV transmission, and informed the application of HIV molecular surveillance in practice.