We have developed new specific primers for sex determination from forensic samples of wolves (Canis lupus), such as hair, saliva, faecal, tooth and urine samples. In order to improve molecular sexing, we performed a multiplex semi-nested polymerase chain reaction (PCR) and several replicated amplifications per sample to avoid errors in low quantity DNA samples, such as allelic dropout and false alleles. The sex of individuals is automatically determined by capillary electrophoresis with a fluorescently labelled internal sex-specific primer from each pair. Our method yielded sex identification on 100% of invasive samples and 93% of forensic samples, being one of the highest success rates obtained from wild animals.