The aims of this study were to compare the performance characteristics and cost-effectiveness of pooling endocervical samples for screening and diagnosis of Chlamydia trachomatis, and to investigate the prevalence of C. trachomatis infection in women in Leningrad Oblast, Russia. A total of 1500 endocervical samples were tested individually and when pooled in groups of 5 and 10 samples, respectively. A previously evaluated in-house diagnostic polymerase chain reaction (PCR) assay was utilized. The sensitivity and specificity of the PCR were not affected by either pooling strategy. The estimated prevalence of genital C. trachomatis infection was 6.6%, 6.1% and 6.0% based on individually tested samples, and pools of 5 and 10, respectively. For diagnosis of individual samples, the pooling strategies resulted in cost savings of 53.3% (5 samples per pool) and 44.0% (10 samples per pool). Pooling samples for PCR detection of C. trachomatis is an accurate and cost-saving approach for diagnosis and large-scale prevalence studies in St Petersburg, Russia.