Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) is considered as the main cause of invasive cervical cancer and cervical intra-epithelial neoplasia. High risk HPV DNA has been shown to be present in 99.7% of cervical cancers. So HPV DNA testing for screening of cervical cancers may play a potential role in early detection and management of cervical cancer. With above background a cross sectional study was undertaken to estimate the prevalence and to identify the associated risk factors of human Papillomavirus infection among Visual Inspection with Acetic acid (VIA) positive women attending at colposcopy clinic of Mymensingh Medical College Hospital (MMCH), Mymensingh, Bangladesh from July 2013 to December 2014. One hundred and forty three cervical swabs for nested PCR were collected from the patients attending colposcopy clinic of MMCH, for detecting target gene of L1 region of the HPV genome. Among the 143 VIA positive patient nested PCR showed 49.6% (71/143) positive. Biopsy of 54 colposcopy positive women revealed that 16 (29.6%) cases were chronic cervicitis, 33 (61.1%) cases were mild dysplasia (C1NI), 01 (1.9%) were having moderate dysplasia (C1NII) and 04(7.4%) patients were diagnosed as invasive squamous cell carcinoma. So, high grade cervical lesions were 100% positive by nested PCR for HPV.