mRNA profiling is a promising new method for the identification of body fluids from biological stains. In this study we aimed to establish a multiplex RT-PCR protocol for the detection and differentiation of sperm and seminal plasma. The Agilent Bioanalyzer and Nanodrop spectrophotometer were shown not to be suitable for assessing RNA quality and quantity of forensic stains. Semen specificity of the mRNA markers was successfully confirmed with singleplex PCR. Our data indicated that semen samples down to 0.1 ml and up to 20-year-old could be identified with mRNA profiling. With the semen multiplex, including 2 sperm markers (PRM1, PRM2) and 2 novel seminal plasma markers (SEMG1, PSA), samples from azoospermic men (absence of sperm in semen) are clearly distinguishable from those of normozoospermic men (having a normal sperm production). We think that our multiplex RT-PCR protocol is a reliable and sensitive method for the identification of semen in forensic samples.