The Eocene alkaline magmatism in the southern Lhasa subterrane provides insights into the timing and dynamic mechanism of the Neo-Tethyan oceanic slab breakoff. Here, we present a combined study of whole-rock geochemistry, zircon U–Pb ages and Sr–Nd–Hf isotopes for the Mayum alkaline complex in the southern Lhasa subterrane. The complex is composed of amphibole syenites, quartz syenite, alkali feldspar granites and mafic microgranular enclaves (MMEs) in the host quartz syenites. Their crystallization ages are 52.2 ± 0.8 Ma, 52.2 ± 1.0 Ma, 49.6 ± 0.5 Ma and 53.1 ± 1.6 Ma, respectively, indicating that these rocks were simultaneously generated in the early Eocene. The hosted alkaline rocks are shoshonic with high SiO2 (64.32–70.49 wt.%), Na2O+K2O (6.63–9.43 wt.%) and low MgO (0.57–2.47 wt.%) contents. They are enriched in large-ion lithophile elements (LILEs: e.g., Rb, K), light rare earth elements (LREEs), Th and U, and are depleted in high field-strength elements (HFSEs) (such as Nb, Ta, and Ti) with strongly negative Eu anomalies (Eu/Eu* = 0.33–0.75). They have homogeneous whole-rock (87Sr/86Sr)i ratios (0.7051–0.7056), low εNd(t) values (–1.7 to –0.9), and positive zircon εHf(t) values (+1.69 to +7.72). Thus, the host alkaline rocks were generated from partial melting of the juvenile lower crust under the southern Lhasa subterrane, and went through a fractional crystallization process during magma ascending emplacement. Compared with the host alkaline complex, the MMEs have lower SiO2 contents (55.16–63.67 wt.%), very higher Fe2O3T (4.08–6.58 wt.%) contents with variable Cr (3.45–226.00 ppm), V (46–107 ppm) and Ni (2.66–6.16 ppm) contents. Meanwhile, the MMEs have similar patterns in trace and rare earth elements (such as obviously Eu anomalies, Eu/Eu* = 0.24–0.38) and similar whole-rock Sr-Nd isotopes ((87Sr/86Sr)i = 0.7051–0.7056, εNd(t) = –2.1 to –1.4) with the host alkaline rocks. Thus, the MMEs might be derived from a mantle source, and magma mixing occurred between the MMEs and the host alkaline rocks. The Mayum alkaline rocks and associated MMEs were generated under an extensional tectonic setting related to the Neo-Tethyan oceanic slab breakoff at ca. 53 Ma.