Non-invasively collected samples such as sloughed skin has proven viable for genetic analysis using microsatellites and mitochondrial DNA, even when degraded. Next-generation sequencing techniques using single nucleotide polymorphisms (SNPs), offer a favourable alternative for degraded DNA, advancing our capacity to address important evolutionary, ecological and conservation questions for long-term studies of globally distributed species. This study utilised a next-generation sequencing approach for the first time to investigate SNP genotyping on 91 humpback whale (Megaptera novaengliae) sloughed skin tissues from both historical (2001 and 2003) and contemporary (2022) collections. DNA quality and quantity assessments confirmed successful long-term preservation and viability of historical skin tissues (>20 years old) with no significant degradation over time. SNP genotyping using sloughed skin produced reliable SNP markers (i.e., high quantity and quality) applicable for future genetic assessments. Three sex-linked markers associated with maleness were identified, suggesting heterogametic male (XX/YY) sex determination, although these markers alone are not reliable for accurate sexing. This study confirms the viability of SNP genotyping from historical and contemporary sloughed skin samples, supporting a collaborative, non-invasive approach to genetic and conservation research on humpback whales and other wide-ranging species.