The objective of the study was to examine the influence of pooling Chlamydia trachomatis specimens. We compared Becton Dickinson ProbeTec strand displacement amplification (SDA) with Cobas Amplicor Roche (PCR). With PCR as the standard, SDA performed equally well in single-sample testing. For pooled PCR samples (compared to individual PCR), we found a sensitivity of 100% and a specificity of 98.9%. For pooled SDA tests (compared to individual SDA), we found a sensitivity of 86.5% and a specificity of 98.9%. Our conclusion is that 2-sucrose phosphate buffer (2-SP) can be used for individual SDA testing. A reduced sensitivity was found for pooled SDA samples, and we cannot recommend that SDA be used for pooling with 2-SP. Further pooling studies with other transport media are warranted.