In order to explore the disputed issue concerning the tectonic affinity of the ancient ocean mantle of North Qilian Mountains (NQM), geochemical and Sr, Nd, Pb isotopic compositions of pillow basalts of the Yushigou Ophiolite (YSGO) suite from NQM have been analyzed systematically. The pillow basalts exhibit tholeiitic characteristics, with flat chondrite-normalized REE patterns ((La/Yb) N = 0.98―1.27). They display no Nb, Ta, Zr, Hf negative anomalies, and show MORB features in 2Nb-Zr/4-Y and Ti/100-Zr-Y×3 tectonic discrimination diagrams. These results indicate that the Yushigou ophiolite is most likely to be formed in a mid-ocean ridge or mature back-arc basin. Their isotopic data show a relatively broad and enriched 87 Sr/ 86 Sr (0.70509―0.70700), restricted 143 Nd/ 144 Nd (0.512955―0.512978). Pb isotopes are in the range of 206 Pb/ 204 Pb (18.054―20.562), 207 Pb/ 204 Pb (15.537―15.743) and 208 Pb/ 204 Pb (38.068―38.530). These isotopic data imply that the basalts originated from the depleted mantle (DMM), with the involvement of enriched mantle components (mainly EMII). Geochemical comparisons between the basalts in YSGO and the MORB-type basalts of ophiolite suites occurring in the known ancient Tethyan tectonic domain indicate that the ancient oceanic mantle represented by YSGO suite forming in early Paleozoic in the North Qilian Moutains is very similar to the Tethyan mantle in both trace elements and isotopic compositions. The North Qilian Mountains should be a part of the Tethyan tectonic domain in early Paleozoic. This further implies that the Tethyan tectonic domain can be deduced to early Paleozoic in the study area, which will be helpful to discussing the tectonic affinity and evolution of the North Qilian Mountains.