OBJECTIVE Clinically, an unbefitting management for high grade squamous intraepithelial lesion (HSIL) may result from an inaccurate diagnosis by colposcopy bioposy.The study aimed to assess the diagnostic accuracy by colposcopic biopsy and evaluate the associated factors in diagnosing HSIL. METHODS Clinical data of 1901 women who were primarily diagnosed as HSIL by colposcopic biopsy and then underwent definitive surgery within six-month interval in Women's Hospital, School of Medicine, Zhejiang University during 2009-2015, were retrospectively collected. The diagnostic accuracy of HSIL by colposcopic biopsy was assessed and the correlations between diagnostic accuracy and clinic-pathological variables were calculated by univariate and multivariate analysis using the pathological diagnosis by definitive surgery as a reference standard. RESULTS The accordance rate of HSIL diagnosis between colposcopic biopsy and definitive surgery was 80.6%, with an under-diagnosis rate of 5.8% and an over-diagnosis rate of 13.6%. Cytology≤low grade squamous intraepithelial lesion(LSIL) (OR:1.599;95%CI:1.185-2.160), colposcopy≤LSIL (OR:2.083;95%CI:1.537-2.824), endocervical curettage (ECC)≤LSIL(OR:2.813;95%CI:2.051-3.857), and lesion without gland involved (OR:1.751;95%CI:1.299-2.361) were independent risk factors for over-diagnosis of HSIL. Women with≥3 risk factors had a 5.078-flod higher risk for over-diagnosis of HSIL compared to those with≤1 risk factor. Irregular vaginal bleeding (OR:2.570,95%CI:1.668-3.960), colposcopy=HSIL (OR:1.699,95%CI:1.022-2.824), ECC=HSIL (OR:2.666, 95%CI:1.728-4.113), and multiple biopsies (OR:1.818, 95%CI:1.153-2.868) were independent risk factors for under-diagnosis of HSIL. Women with ≥3 risk factors had a 5.710-flod higher risk for under-diagnosis of HSIL compared to those with ≤1 risk factor. CONCLUSIONS The diagnostic accuracy of HSIL by colposcopic biopsy is about 80% and associated with some factors including symptom, cytology result, colposcopy diagnosis, and biopsy number. These variables may be predictors for over-diagnosis or under-diagnosis of HSIL by colposcopic biopsy.