Understanding shark reproductive modes is fundamental for their conservation, as these K-strategist species are highly susceptible to overexploitation. The spiny dogfish ( Squalus acanthias), a small shark currently listed as ’vulnerable’ by the IUCN, exhibits a prolonged gestation period of 22 months and a reproductive output ranging from 1 to 21 pups per litter. This study aimed to investigate the occurrence of multiple paternity in S. acanthias using Single Nucleotide Polymorphism (SNP) markers. Samples from six litters, comprising 40 individuals collected in Argentina, were analyzed using a ddRADSeq library. SNP markers were screened through the STACKS pipeline, and kinship and paternity analyses were conducted using COANCESTRY and COLONY softwares. The results revealed between 1,021 and 1,620 SNPs per litter, with multiple paternity detected in all litters. The number of sires per litter ranged from 2 to 4. Notably, no correlation was found between litter size and the occurrence of multiple paternity, suggesting that this behavior may represent a reproductive strategy aimed at enhancing genetic diversity. The species’ biological characteristics, such as size and sex segregation, combined with the presence of females in shallower waters, increase their susceptibility to capture. Moreover, overfishing and bycatch in the Atlantic exacerbate the reduction in sexually mature individuals, thereby threatening reproductive rates. This study underscores the importance of preserving genetic variability for species survival and highlights the need for urgent management policies that incorporate these reproductive strategies, particularly for species like S. acanthias that display complex life histories and low population recovery rates.