Few studies have delimited evolutionary entities within the Sombre Tit, Poecile lugubris. Here, we explored its morphological and genetic variability using 24 morphometric variables, two mitochondrial (COX1 and ND2), two nuclear (ODC and MB), and 10 microsatellite loci. Genetic and morphometric characters supported the species status of the Caspian Tit, Poecile hyrcanus with a separation from P. lugubris more than 4.5 MYA. The phylogenetic analyses uncovered three distinct clades within P. lugubris. The subspecies P. l. lugubris, with strong genetic differences from the other subspecies (4.5%), diverged at ∼1.1 MYA. Samples from Iran formed the remaining two clades. Individuals from western and northwestern Iran were placed in a single clade (anatoli), while those from southern and southwestern Iran were in another independent clade (dubius-kirmanensis). Morphometric analyses also confirmed this pattern. The microsatellite results discriminated Iranian subspecies as discrete clusters with signs of nuclear admixture between dubius and anatoli in the Zagros Mountains. Signs of population expansion for anatoli and dubius-kirmanensis coincided with the late LGM. Our results shed new light on the phylogenetic relationships, evolutionary history, and past demographic processes of P. lugubris.

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