Freshwater populations of typically marine species present unique opportunities to investigate biodiversity, evolutionary divergence, and the adaptive potential and niche width of species. A few pinniped species have populations that reside solely in freshwater. The harbour seals inhabiting Iliamna Lake, Alaska constitute one such population. Their remoteness, however, has long hindered scientific inquiry. We used DNA from seal scat and tissue samples provided by Indigenous hunters to screen for mitochondrial DNA and microsatellite variation within Iliamna Lake and eight regions across the Pacific Ocean. The Iliamna seals (i) were substantially and significantly discrete from all other populations (
x
¯
F
st-mtDNA
= 0.544,
x
¯
Φ
st
-
mtDNA
= 0.541,
x
¯
F
st-microsatellites
= 0.308), (ii) formed a discrete genetic cluster separate from all marine populations (modal ∆
k
= 2, PC1 = 14.8%), had (iii) less genetic diversity (Hd,
π
,
H
exp
), and (iv) higher inbreeding (
F
) than marine populations. These findings are both striking and unexpected revealing that Iliamna seals have likely been on a separate evolutionary trajectory for some time and may represent a unique evolutionary legacy for the species. Attention must now be given to the selective processes driving evolutionary divergence from harbour seals in marine habitats and to ensuring the future of the Iliamna seal.