Objective: To evaluate the effects of infection in multiple types of high-risk human papilloma virus (HPV) in cervical preneoplastic lesions in patients undergoing colposcopy following a diagnosis of atypical squamous cells of unknown significance (ASCUS) and low-grade squamous intraepithelial (LSIL) cytology. Materials and Methods: Between 2009 and 2010, 2,500 patients were recruited with a mean age of 35 +/- 5 years. Screening for cervical cancer was performed and in case of ASCUS and LSIL the patients underwent colposcopy. The tests for the detection and typing of viral DNA (HPV - DNA test) were performed on cervical swab with real-time PCR amplification. Results: The prevalence of infection was 70% (1579/2256) in the patients recruited. In relation to the degree of preneoplastic lesions some high-risk HPV viral genotypes were identified: HPV 16 (319/1466), HPV 18 (164/1466). HPV 45 (76/1466), HPV 31 (215/1466), HPV 52 (145/1466), HPV 58 (55/1466) HPV 56 (79/1466), HPV 51 (110/1466), HPV 6 (138/1466), HPV 11 (88/1466), HPV 42 (34/1466), HPV 53 (43/1466). In case of high-grade lesions of CIN (CIN2 and CIN3) a greater HPV co-infection was detected and in particular the association from 16 to 18 (70%), 16-33 (18%) and 16 to 52 (12%). Conclusions: Infection caused by the simultaneous presence of multiple HPV genotypes appears to be associated with a significantly increased risk of high-grade lesions of CIN or invasive cancer than the presence of single viral infections. The infection with multiple HPV types is a significant risk factor for high-grade lesions of CIN in women undergoing colposcopy for ASCUS cytology / LSIL. The use of real-time PCR has shown the ability not only to identify the different types of HPV, but also to monitor quantitatively the same over time, and during the study phase, to evaluate the sensitivity and specificity of the method in comparison with other techniques.