The broom hare (Lepus castroviejoi) is a threatened Iberian endemic, for which there is limited knowledge. We use genetic non-invasive sampling (gNIS; N = 185 faeces samples) and specimens from hunting and roadkills (N = 22) in conjunction with a 15-microsatellite panel and a 541-bp fragment of cytochrome-b to assess the genetic diversity, population structure and evolutionary history of this species. Populations from the other four European hare species were also analysed to accurately compare the genetic diversity patterns and infer admixture. Species identification from gNIS was inferred using small fragments of cytochrome-b and transferrin genes and individual identification was obtained using microsatellites. The broom hare population showed the lowest level of nuclear DNA diversity of all analysed hare species (N = 76; Na = 2.53, He = 0.186 and Fis = 0.341) and very low mitochondrial DNA diversity (N = 64; Hd = 0.743 and π = 0.01543). Only the Italian hare (L. corsicanus) showed a similar pattern of low genetic diversity. No hybridization with the neighbouring hare species was detected. However, two mitochondrial DNA lineages, corresponding to two ancient events of introgression of mountain hare (L. timidus) origin, were characterized. There was evidence for shallow spatial population differentiation of the broom hare. The described reduced genetic diversity, associated with a narrow distribution range and recent population declines, represents a risk of population extinction, and highlights the need for conservation measures of this endemic threatened hare species.