Abstract Background In the Netherlands, insight into sexually transmitted infection (STI) testing and characteristics of those tested by general practitioners (GPs) and sexual health centres (SHC) is limited. This is partly due to lacking registration of socio-demographics at GPs. We aimed to fill this gap by linking different registers. Methods Individual STI testing data of GPs and SHC were linked to population register data (aged ≥15 years, Rotterdam area, 2015–2019). We reported population-specific STI positivity, proportion STI tested, and GP-SHC testing rate comparison using negative binomial generalised additive models. Factors associated with STI testing were determined by the provider using logistic regression analyses with generalised estimating equations. Results The proportion of STI tested was 2.8% for all residents and up to 9.8% for younger and defined migrant groups. STI positivity differed greatly by subgroup and provider (3.0–35.3%). Overall, GPs performed 3 times more STI tests than the SHC. The smallest difference in GP-SHC testing rate was for 20–24-year-olds (SHC key group). Younger age, non-western migratory background, lower household income, living more urbanised, and closer to a testing site were associated with STI testing by either GP or SHC. GPs and SHC partly test different groups: GPs test women and lower-educated more often, the SHC men and middle/higher educated. Conclusions This study highlights GPs’ important role in STI testing. The GPs’ role in the prevention, diagnosis, and treatment of STIs needs continued support and strengthening. Inter-professional exchange and collaboration between GP and SHC is warranted to reach vulnerable groups.