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Molecular sexing is routinely used in the fields of forensic investigations, population genetics and conservation biology. However, none of the assays used so far allows non-ambiguous, quick, and cheap sex identification in snakes. Here, we designed a new multiplex PCR (two panels of triplex) sexing assay using two homologous loci on sex chromosomes (snake gametologous genes), CTNNB1Z/CTNNB1W and WACZ/WACW, and two combined female-specific CTNNB1W loci. This method was successfully tested on 81 samples from 17 caenophidian snakes, but not for 20 samples from 5 henophidian snakes, suggesting that this assay was applicable across all caenophidian snake tested. Moreover, the results of the validation study comprising sensitivity, specificity, blind test, and casework with blood spots on filter paper and shed skin as a non-invasive sample confirmed the effectiveness of this assay in the forensic perspective. The assay represents a robust, cheap, rapid, and simple method which should also prove useful in unknown bodies of carcass. This efficient assay should benefit wildlife forensic laboratories and registered breeders to conform to wildlife regulations or certification of commercial trade, and boost profits for snake-breeders and conservationist snake-breeding communities.