The organic matter-rich mudstones of the Middle Jurassic Dameigou Formation in the northern Qaidam Basin are regarded as the principal petroleum source rocks for unconventional resource exploration in China. In this study, a detailed investigation on the organic petrography, inorganic geochemistry as well as bulk and molecular organic geochemistry is presented. Three different source rock units were identified with TOC contents and VRr ranging from 1.5–39.2% and 0.35–0.57%, respectively. Carbonaceous mudstone and organic matter-rich mudstone in unit 1 with high Pr/Ph (4.79–7.25) and low Gam index (0.02–0.03) values mainly developed in an oxic freshwater column under warm and humid climatic conditions and strong weathering intensity. Organic matter in unit 1 with high proportions of C29 ααα 20R steranes (61–74%) is interpreted to be derived from terrestrial higher plants. By contrast, units 2 (Pr/Ph: 2.56–4.23; Gam index: 0.03–0.14) and 3 (Pr/Ph: 0.73; Gam index: 0.48) were deposited in sub-oxic to oxic freshwater and anoxic saline environments, respectively. Progressive aridification during the deposition of units 2 and 3 are reflected by decreasing CIA values (59.97–86.68) resulting in weakened weathering intensity and lower terrestrial influx. A mixture of terrestrial higher plants and algal biomass was the main source of organic matter in unit 2 with moderate C19/C23 Tri (0.96–5.44), C20/C23 Tri (1.28–3.87) and C24 Tet/C26 Tri (1.10–2.54) ratios, while unit 3 with the lowest C19/C23 Tri (0.25), C20/C23 Tri (0.95), and C24 Tet/C26 Tri (0.65) ratios and highest proportions of C27 ααα 20R steranes (69%) is characterized by a decrease in terrigenous organic matter content. The dominant source of organic matter is lake plankton, including halophilic algae and cyanobacteria. Organic matter accumulation in unit 1 is associated with a warm and humid climate, weathering intensity, and terrigenous organic matter input. Primary productivity is the most important factor controlling the enrichment of organic matter in units 2 and 3.