DNA fingerprinting is one of the most reliable techniques in forensics with highest possible accuracy and detection limits. The results are crucial to link the suspect to the crime. However it is observed that delay in medical examination prior to the collection of biological samples of victim for DNA fingerprinting reduces the chances of semen, DNA isolation from various exhibits and subsequent detection. A comparative study of analysis of different types of biological samples has been carried out to find the best source for semen detection among the samples collected from the victim. A total of 200 different samples such as swabs from different parts of victim’s body, clothing and vaginal wash were examined for the presence of semen based on presumptive tests and microscopic examination followed by DNA fingerprinting. Study shows vaginal wash as best source with respect to uncontaminated semen and quantity of DNA which enable to generate accurate DNA profile.