Objective: The aim of this study was to explore a panel of useful markers in differential diagnosis of primary endocervical adenocarcinoma (ECA) and endometrial adenocarcinoma (EMA). Materials and Methods: Thirty-three ECAs and 31 EMAs were collected and examined for high-risk human papillomavirus (hr-HPV) (16/18) DNA using in situ hybridization, and for p16, vimentin, ER, PR expression using immunohistochemistry (IHC). Results: Detection rate of hr-HPV (16/18) DNA in ECA (72.7%, 24/33) was significantly higher than that in EMA (12.9%, 4/31) (p < 0.01). Twenty-four of 33 (72.7%) cases of ECA, but only five of 31 (16.1%) cases of EMA showed high expression of p16. Twenty-three of 24 (95.8%) hr-HPV DNA-positive ECA and all four (100.0%) hr-HPV DNA-positive EMA showed high levels of p16 expression. High expression rates of vimentin (90.3%, 28/31), ER (58.1%, 18/31), and PR (71.0%, 22/31) in EMA were significantly higher than those in ECA, respectively (p < 0.01). Conclusion: Detection of hr-HPV DNA combined with immunohistochemical expressions of p16, vimentin, ER, and PR have important value in differential diagnosis between ECA and EMA.