Human papillomavirus (HPV) plays an important role in the pathogenesis of cervical cancer. HPV has been found in 99.7% of cervical cancers worldwide. In Malaysia, it is the second most common cancer among women in all major ethnic groups. The main purpose of this study was to establish the method of SyBrGreen Real-Time PCR and apply it for identification of multiple infections of the two high risk HPV subtypes. In this study, 57 positive samples for HPV 16 and HPV 18 were used to establish a simple and sensitive method to detect and identify HPV infection in the cervical neoplasia at different stages of the disease by using real-time ABICycler SyBrGreen 1 technology. The results showed 67 HPV genomes in 57 samples. HPV 16 genome was detected in 55/67 (82%) cases while HPV 18 was detected in 8/67 (12%) cases with 4 cases showing multiple infections of HPV 16 and HPV 18. HPV 16 was the most prevalent followed by HPV 18. Using SyBr Green Real-Time PCR techniques, the results showed that DNA melting curve for HPV 16 had a peak around 80.2ºC and Ct value of 20 cycles whereas the DNA melting curve for HPV 18 around 79.2ºC and Ct INTRODUCTION value of 22 cycles. In conclusion, a SyBr Green Real-Time PCR method has the potential for clinical usage in detection and identification of HPV infection in cervical neoplasia at different stages of the disease.