This study is a critical analysis of certain amplification assays for detecting Chlamydia trachomatis and Neisseria gonorrhoeae infections which have demonstrated that the plasmid-free variant of C. trachomatis is frequently responsible for infection in our patients. Specifically, we evaluated the performance of the strand displacement amplification (SDA) assay in detecting either C. trachomatis or N. gonorrhoeae in 1,190 clinical samples, both urogenital and ocular, from 1,005 consecutive patients. The results obtained with the BDProbeTec ET System were compared with three referenced amplification methods for C. trachomatis (detecting the 16S rRNA gene, the omp1 gene and the plasmid of C. trachomatis) and with both the culture method as well as an amplification assay followed by genetic identification performed using the MicroSeq 500 16S ribosomal DNA-based system for N. gonorrhoeae. The sensitivity of SDA (76%) in detecting C. trachomatis is significantly low when compared with that of other molecular techniques employing 16S rDNA or omp1 as a target. The specificity of the methods for detecting C. trachomatis was excellent, ranging from 99.4 to 100%. Furthermore, the results of SDA in detecting N. gonorrhoeae also provided excellent results (100% specificity and sensitivity).