Abstract Background It is important to understand how HIV infection is transmitted in the population in order to guide prevention activities and properly allocate limited resources. In Ukraine and other countries where injecting drug use and homosexuality are stigmatized, the information about mode of transmission in case registration systems is often biased. Methods We conducted a cross-sectional survey in a random sample of patients registered at HIV clinics in seven regions of Ukraine in 2013–2015. The survey assessed behavioral risk factors and serological markers of viral hepatitis B and C. We analyzed the discrepancies between the registered mode of transmission and the survey data, and evaluated trends over 3 years. Results Of 2285 participants, 1032 (45.2%) were females. The proportion of new HIV cases likely caused by injecting drug use based on the survey data was 59.7% compared to 33.2% in official reporting, and proportion of cases likely acquired through homosexual transmission was 3.8% compared to 2.8%. We found a significant decrease from 63.2 to 57.5% in the proportion of injecting drug use-related cases and a steep increase from 2.5 to 5.2% in homosexual transmission over 3 years. Conclusions The study confirmed the significant degree of misclassification of HIV mode of transmission among registered cases. The role of injecting drug use in HIV transmission is gradually decreasing, but remains high. The proportion of cases related to homosexual transmission is relatively modest, but is rapidly increasing, especially in younger men. Improvements in ascertaining the risk factor information are essential to monitor the epidemic and to guide programmatic response.