The aims of this study were to compare performance characteristics and cost-effectiveness of pooling urine samples for screening and diagnosis of Chlamydia trachomatis using Digene Hybrid Capture II CT/NG Test (HCII), and to examine the prevalence of C. trachomatis in male military recruits in Lithuania. A total of 410 urine samples were individually tested and pooled by 5 and 10 samples, respectively. The sensitivity and specificity of diagnosis were not affected by either pooling strategy. The estimated population prevalence of C. trachomatis infection was nearly identical, i.e. 4.4%, 4.4% and 4.1% based on individually tested samples, and samples pooled by 5 and 10, respectively. For this estimation of the population prevalence, pooling 5 samples reduced the costs by 80% and pooling 10 samples reduced the costs by 90%. For diagnosis of each individual sample, the pooling strategies resulted in cost savings of 60% (5 samples per pool) and 56% (10 samples per pool). The present pooling strategies were sensitive, specific and cost-efficient for screening and diagnosis of C. trachomatis infection in male military recruits in Lithuania. The strategies would be most useful for reasonably inexpensive large-scale screening, prevalence studies and even diagnostics in Lithuania and many other low-resource countries.