We conducted a tricenter interlaboratory agreement study to assess the agreement of PCR results obtained for detection of Chlamydia trachomatis in genitourinary specimens. A total of 120 specimens (49 positive and 71 negative), including 20 first-void urine samples, 50 endocervical and 50 urethral swabs (40 males), were coded and sent from a reference laboratory (laboratory A) to two other laboratories. Laboratories B and C were provided with a standardized protocol and reagent package including two sets of plasmid PCR primers (KL1-KL2 and T1-T2) and were asked to test each specimen with the first set of primers (KL1-KL2) and to confirm positives with the second set of primers (T1-T2). Laboratory B identified 47 of 49 positives and 69 of 70 negatives (one specimen dried up on shipping) following the initial PCR, for an accuracy of 97.5% (116 of 119), and 47 of 49 positives and 70 of 70 negatives after confirmatory testing of the positives, for an accuracy of 98.3% (117 of 119). Laboratory C identified 42 of 49 positives and 70 of 70 negatives for the initial PCR, for an accuracy of 94.1% (112 of 119), and 39 of 42 positives and 70 of 70 negatives for the confirmatory PCR, for an accuracy of 91.6% (109 of 119). The overall accuracy of PCR testing was 96.6% (345 of 357). The kappa agreement statistics for agreement between pairs of laboratories after confirmation of positives were 0.97 for laboratories A and B, 0.83 for laboratories B and C, and 0.83 for laboratories A and C. Use of the confirmatory PCR improved the specificity and overall accuracy of results for individual laboratories but reduced slightly the results obtained for agreement between laboratories. These results demonstrate that when standardized reagents and protocols are used, PCR results are highly reproducible and excellent agreement between laboratories is obtainable.