Lath martensite substructures in as-quenched plain carbon steels exhibit dislocation-like contrast in the transmission electron microscope. More recent observations reported internal twins and nanoscale auto-tempered intra-lath carbides as additional lath substructures in ultra-low-C binary Fe–C steels. Modern microalloyed steels often have similar ultra-low C contents besides microalloying elements like Ti, Nb or V and, more recently, Mo, to achieve high strength, toughness and weldability. Nonetheless, little is known about the lath substructure evolution in the as-quenched state of microalloyed steels. This study investigates the hierarchical martensite substructure evolution post-quenching of microalloyed Nb and NbMo steels with 0.1 wt% C. Hierarchical microstructure characterization was done using scanning and transmission electron microscopy, and electron backscatter diffraction methods including parent grain reconstructions with MTEX. Thermokinetic simulations using MatCalc to determine the carbide evolution during auto-tempering were corroborated with site-specific transmission electron microscopy. Mo addition led to lowering of the martensite start temperature, yet the Nb steel showed a finer hierarchical microstructure. Finer laths with in-lath dislocations, short and long twins, and lath boundary decoration of carbides were found in the Nb steel. Conversely, laths in the NbMo were wider, with frequent intra-lath auto-tempered precipitates in the vicinity of dislocations, without twins.