BACKGROUND: This study evaluated the intra- and inter-observer reproducibility of the dual-stain biomarker, CINtec® PLUS cytology in ThinPrep® specimens, for improved specificity in the detection of cervical disease in women testing human papillomavirus (HPV) positive. METHODS: A total of 972 cases of HPV-positive women from a triage and primary HPV screening population were selected from an ongoing study evaluating the clinical performance of CINtec® PLUS cytology. For reproducibility analyses, three cytotechnologists rescreened sets of slides which they had previously reported themselves and which were previously reported by each of the other cytotechnologists. The original results of slides previously screened by each of the three cytotechnologists were also compared with the results of an expert reference evaluator. RESULTS: Intra- and inter-observer agreement for paired evaluations between reviewers ranged from 82.8% to 94.9% (kappa 0.65-0.91) and 89.2% to 93% (kappa 0.83-0.88), respectively. Reproducibility analyses between the cytotechnologists and the reference evaluator revealed agreements ranging from 95.5% to 98% (kappa 0.89-0.96). CONCLUSION: Evaluation of the dual-stain biomarker showed a high level of agreement across all evaluators suggesting that CINtec® PLUS cytology will perform well in the hands of cytotechnologists and pathologist reviewers and could be introduced into cellular pathology laboratories that employ ThinPrep® LBC with a minimum effort.