Population genetics is essential for understanding and managing marine ecosystems, but sampling remains challenging. We demonstrate that high-throughput sequencing of seawater environmental DNA can provide useful estimates of genetic diversity in a whale shark (Rhincodontypus) aggregation. We recover similar mitochondrial haplotype frequencies in seawater compared to tissue samples, reliably placing the studied aggregation in a global genetic context and expanding the applications of environmental DNA to encompass population genetics of aquatic organisms.