In Iran, the Elburz and Zagros Mountain ranges include substantial karst regions housing numerous aquifers and groundwater resources. Niphargus Schiødte, 1849, a diverse subterranean amphipod genus, inhabits Western Palearctic groundwater environments, with Iran marking the eastern limit of its distribution. This study examined specimens collected from springs along the Elburz and Zagros Mountains, revealing two distinct taxonomic units through a combination of morphological observations and molecular analyses, utilizing COI and 28S rDNA genes. N. elburzensissp. nov. is characterized by produced epimeral plates I to III; a telson lobe with five distal, two lateral, and one mesial spine each, a rectangular shape of gnathopod II propodi with two L-setae on palmar corner and maxilla I outer plate spines with 2-2-1-1-3-0-1 denticles. N. zagrosensissp. nov. is distinguished by a triangular shape gnathopod II propodi, pereopod VI longer than pereopod VII, maxilliped outer plate less than half of palp article 2, and uropod III distal article exceeding 80% of the proximal article. Pairwise genetic distances between N. elburzensissp. nov. and other species ranged from 10.70% (N. fiseri) to 23.48% (N. daniali) for COI gene and 1.56% (N. urmiensis) to 10.98% (N. daniali) for 28S gene. Also, N. zagrosensissp. nov. exhibited COI gene distances from 5.73% (N. alisadri) to 20.66% (N. daniali) and from 0.13% (N. alisadri) to 11.36% (N. daniali) for 28S gene distances. Bayesian analysis suggests that the two newly discovered species are part of the expansive local Iranian clade. These species are supported phylogenetically by separate and independent lineages, as indicated by high bootstrapping values.