In a criminal investigation, it is important to clarify the origin of the extracted DNA from available biological samples. In recent years, a new approach for the identification of body fluid stains has been demonstrated and involves a comparison of specific mRNA expression levels. Here, we used real-time RT-PCR to examine the expression levels of STATH , HTN3 , MUC4 , and ESR1 genes from salivary, nasal, and vaginal secretions which are often thought to be difficult to discriminate between. STATH was expressed at high levels in all salivary (dCt=1.27±3.18, n =10) and all nasal secretions (dCt=−0.99±3.94, n =8), and at low levels in 3 of the 10 vaginal secretions (dCt=16.34−17.47). HTN3 was expressed at high levels in all salivary secretions (dCt=0.67±3.08). MUC4 was expressed at relatively high levels in all vaginal secretions (dCt=4.97±2.36, n =10), and at moderate levels in 5 of the 10 salivary secretions (dCt=8.55−10.40) and in 4 of the 8 nasal secretions (dCt=6.56−8.23). ESR1 was expressed at relatively high levels in all vaginal secretions (dCt=5.79±2.90), but no expression was found in salivary and nasal samples. These results indicate the possibility for specific discrimination between various body fluids. To test the practicality of this, gene sensitivity and stability were examined in saliva, semen, and blood stains. Several genes, including ACTB , STATH , HTN3 , PRM2 , SEMG1 , and HBB , were shown to be detectable even in small volume (0.1 μL) stains and were also expressed in 1-year-old stains. mRNA stability was dependent on favorable environmental factors, such as dryness and shading. In addition, the successful detection of target genes from mixed stains and simulated casework samples might promise the utility of this assay. The mRNA assay therefore appears to be a novel tool for body fluid identification from biological samples.