BackgroundRegular HIV testing is the best way to detect people living with HIV promptly, yet not much is known about the characteristics of frequent, voluntary testers. This study explores factors related to HIV testing frequency among five key populations in China including men who have sex with men (MSM), female sex workers (FSWs), people who use drugs (PWUD), men who have casual sex with women (MCSW) and sero-negative partners among sero-discordant couples (SNPs).MethodsWe conducted a cross-sectional study in ten cities of China from November 2018 to September 2019 using convenience sampling to recruit participants. Univariate and multivariate partial proportional odds models were adopted to compare socio-behavioral factors associated with HIV testing frequencies among the five key populations.ResultsAmong the 2022 recruited participants, 36.6% reported not testing for HIV in the past year, whereas 37.0% tested once and 26.4% tested twice. Compared with MSM, FSWs (AOR = 1.97, 95% CI: 1.36–2.86) and SNPs (AOR = 3.63, 95% CI: 2.40–5.49) were more likely to test for HIV, but MCSW (AOR = 0.23, 95% CI: 0.17–0.32) were less likely. Additionally, SNPs (AOR = 4.02, 95% CI: 2.78–5.83) were more likely to be frequent HIV testers, while FSWs (AOR = 0.49, 95% CI: 0.32–0.76) and MCSW (AOR = 0.29, 95% CI: 0.20–0.41) were less likely to be frequent testers. Factors identified as barriers to HIV testing include the following: higher education level and > 5000 CNY monthly income for FSWs; elder age and a married/cohabitating status for PWUD; reported alcohol use for MCSW; and non-Han ethnicity and non-local household for SNPs. Facilitators to frequent testing included the following: higher education level for MSM and SNPs; higher AIDS knowledge score for MSM and PWUD; > 5000 CNY monthly income for FSWs and PWUD; and reporting high-risk sexual behaviors for MSM, FSW and PWUD.ConclusionsHIV testing frequencies and associated factors were not equivalent across the five key populations in China. Public health officials should take heed of the identified high-risk populations reporting high testing rates, perhaps with intensive and tailored behavioral interventions or biochemical prophylaxis.