Genetic sex identification and efficient microsatellite genotyping using non-invasive samples are important for assessing genetic diversity and structure of wild mammals. We extracted DNA from fecal samples of endangered proboscis monkeys (Nasalis larvatus) at the Yokohama Zoological Gardens, and successfully identified their sex using a DEAD-box gene marker. Using the information of 31 published microsatellite loci developed for proboscis monkeys, we redesigned the primers for 18 loci and selected microsatellite loci with high heterozygosities and then developed a multiplex PCR system, in which 19 polymorphic microsatellite loci and a sex identification marker were amplified in three multiplex sets. In our developed method, genotyping errors rarely happened using DNA extracted from feces, and we could identify individuals and estimate parent—offspring relationship with high confidence. Our developed method is applicable for genetic analysis of wild proboscis monkeys and will provide us the information of not only their ecological and social features but also conservation management.