Serology routinely serves as a diagnostic tool to confirm infections in humans. Particularly in delayed settings, such as post-outbreak scenarios where the acute phase of infection has subsided, serology is invaluable. Multiple studies, nonetheless, indicate deficiencies in specificity and sensitivity of current chlamydial antibody detection assays. Incorporation of multiple antigens per target is known to improve the accuracy of chlamydial serological assays. We, therefore, used the recomLine test (Mikrogen diagnostics) on serological samples of two cohorts, as it is the only commercially available test allowing detection of antibodies against three human pathogenic species (, and ) using multiple antigens per target. The first cohort (n = 156; samples collected between 2008 and 2022 during a screening initiative) comprised women from the Netherlands (NL) with past exposure to , while the second cohort (n = 44; samples collected in 2018 in a health examination survey) consisted of Belgian citizens (BE) with occupational or recreational exposure to chickens, representing a risk population for . The test indicated a statistically equivalent seroprevalence in both cohorts (39.10% in NL and 34.09% in BE; p = 0.337). As expected seroprevalence was significantly higher (p < 0.001) in the Dutch cohort (48.72%), as compared to the Belgian cohort (4.55%). Lastly, seroprevalence did not significantly differ between the two groups (2.27% in BE and 1.92% in NL; p = 0.633), even though a higher prevalence was expected for the Belgian cohort. This prompts us to question whether the Belgian cohort truly constituted a risk population or whether the recomLine test is susceptible to cross-reaction of species-specific antibodies, thereby increasing prevalence in the Dutch cohort. We advocate for the development of affordable, highly sensitive antibody detection assays that can effectively distinguish between chlamydial species, addressing the increasing demand for enhanced serological testing methodologies.