Semen stains are the most important biological evidence when identifying the aggressor in sexual assault cases. Current detecting assays of semen stains species identification were not confirmative enough. In this study, we investigated the potential of species identification of semen stains by using attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy combined with advanced chemometrics methods. The effect of substrates types and time since deposition (TSD) were considered in the study. A partial least squares-discriminant analysis (PLS-DA) classification model was established which demonstrated complete separation between human and other species (rabbit, dog, boar, bull, and ram). Validation was conducted which showed prediction abilities with 100% accuracy. Additionally, we found species identification could be achieved without sperm cells which proved ability of spectroscopic methods detecting the semen samples from the case of azoospermia. This work provides a powerful and practical tool for species identification of semen stains in real forensic casework.