For conservation, ethical and welfare reasons, minimally invasive DNA sampling is becoming increasingly important in animal genetics studies. Skin mucus swabs have been used as a source for fish DNA in PCR-based studies, but their suitability for high-throughput sequencing has not been examined. DNA was extracted from skin mucus, muscle and fin samples of two Nile tilapia and used in double-digest restriction-site associated DNA (ddRAD) sequencing. Approximately 16,000 and 9000 RAD loci were retrieved from de novo and reference-based analyses respectively. The numbers of RAD loci retrieved from three tissue sources were similar, with >83 % being shared among all three samples. Minor bacterial contamination was detected in a single muscle sample (0.07 % of the total RAD loci). The data indicates that DNA derived from skin mucus can be reliably used for ddRADseq and is likely to be applicable in other similar genomic analyses.