Abstract Background In Switzerland, HIV-1 transmission among men who have sex with men (MSM) has been dominated by subtype B, whilst non-B subtypes are commonly attributed to infections acquired abroad among heterosexuals. Here, we evaluated the temporal trends of non-B subtypes and the characteristics of molecular transmission clusters (MTCs) among MSM. Methods Sociodemographic and clinical data and partial pol sequences were obtained from participants enrolled in the Swiss HIV Cohort Study. For non-B subtypes, maximum likelihood trees were constructed, from which Swiss MTCs were identified and analyzed by transmission group. Results Non-B subtypes were identified in 8.1% (416/5116) of MSM participants. CRF01_AE was the most prevalent strain (3.5%), followed by subtype A (1.2%), F (1.1%), CRF02_AG (1.1%), C (0.9%), and G (0.3%). Between 1990 and 2019, an increase in the proportion of newly diagnosed individuals (0/123 0% to 11/32 34%) with non-B subtypes in MSM was found. Across all non-B subtypes, the majority of MSM MTCs were European. Larger MTCs were observed for MSM than heterosexuals. Conclusions We found a substantial increase in HIV-1 non-B subtypes among MSM in Switzerland and the occurrence of large MTCs, highlighting the importance of molecular surveillance in guiding public health strategies targeting the HIV-1 epidemic. The proportion of MSM with newly diagnosed HIV-1 non-B subtypes has strongly increased since 1990, reaching 34% of all MSM diagnosed in 2019. This is partly due to ongoing local transmission in this population, as evidenced by large transmission clusters.