Background and rationale: Semen is one of the most common body fluids found at the crime scene. Protamine-1 (PRM1), Protamine-2 (PRM2) and semenogelin-1 (SEMG1) are specific mRNA markers found only in semen so if it is possible to detect these markers in a forensic specimen by RT-PCR, this could be helpful to verify the presence of semen.Aim of the Work:This work aims at identifying semen from other body fluids in forensic samples by molecular assessment of specific RNA biomarkersSubjects and methods:Semen samples were collected from 50 inpatients at the andrology section and outpatients at the andrology clinic that were divided into 3 main groups including: normozospermic, oligozospermic and azoospermic groups and only 10 blood samples were taken from the same patients to assess the presence of the markers in blood. The next steps of work were RNA extraction from semen and blood, PCR amplification of genes, detection of the amplified gene using agarose gel electrophoresis and performing gene expression analysis of the 3 genes using quantitative RT-PCR.Results:(PRM1), (PRM2) and (SEMG1) were only detected in semen samples and were totally absent in blood samples. PRM1 was the most specific and reliable marker for semen identification followed by PRM2 and SEMG1 was the least specific. These markers are not only used for identification but also for detection of the semen profile of the suspect. There was a direct positive correlation between sperm count and RNA expression. Light and humidity are detrimental factors for RNA stability.Conclusion and Recommendations:Each tissue or cell type makes a unique constellation of mRNAs, some specific for only that tissue or cell type. Therefore, analysis of the “RNA profile” in a sample can uniquely identify the fluid or tissue of origin.RT- PCR is valuable in semen identification by gene expression analysis of PRM1, PRM2 and SEMG1. PRM1 is the most specific and reliable marker used for semen identification. Light and humidity are important factors affecting RNA expression.