Blackfin tuna (Thunnus atlanticus) is a small tuna distributed in the western Atlantic Ocean where it is exploited by growing recreational and commercial regional fisheries. In this work, genome-wide genetic variation was analysed to investigate the occurrence of stock subdivision. A de novo assembly of the blackfin tuna genome was generated using Illumina paired-end sequencing data and applied as a reference for population genomic analysis of specimens from nine localities (average sample size per locality n = 72) spanning most of the blackfin tuna distribution range. A total of 2139 single-nucleotide polymorphisms were discovered and genotyped using the double-digest restriction associated DNA sequencing. Pairwise exact homogeneity tests were significant in 24 out of 36 population pairs and significant spatial autocorrelation of genotypes was observed for specimens collected within 2250 km of each other. However, divergence among locality samples was very low (pairwise FST range 0.0002–0.0025) and significant temporal variations were detected in localities sampled multiple times. Approaches to detect cryptic groups de novo were unsuccessful. Additional sampling is warranted to determine if multiple stocks need to be defined for management and assess temporal and spatial patterns of gene flow connecting them.