The uplift of the Qinghai–Tibet Plateau has shaped a unique extreme environment, fostering distinct endemic aquatic organisms. Schizopygopsis pylzovi, a vulnerable endemic fish in the upper Yellow River, is a key model for studying the biogeographic patterns of plateau fish. To assess its genetic characteristics and evolutionary dynamics, we comprehensively evaluated 11 geographic populations of S. pylzovi using two complementary mitochondrial markers, the conserved COI gene and the fast-evolving D-loop region. A total of 142 COI and 143 D-loop sequences were analyzed, and sequences alignment, haplotype network construction, AMOVA, and neutrality tests were performed. AMOVA revealed that genetic variation was mainly distributed within populations, indicating weak population differentiation. Neutrality tests and mismatch distribution analysis suggested historical and recent population expansion events. Our findings highlight the value of joint analysis using COI and D-loop markers in revealing the genetic structure of S. pylzovi, provide new insights into the impact of plateau uplift on fish evolution, and establish a scientific basis for the conservation of this vulnerable species.