Multi-pass welding is used in high-pressure and thick-walled pipes in natural gas and oil pipelines. When a welding layer of a welded joint is subjected to different welding thermal cycles, its microstructure and properties change, thereby affecting the overall welding performance. In this study, the temperature and microstructural variations of the fill pass 2 (FP2) in the entire welding process were investigated by combining the thermal cycle with the cascade welding method. The original FP2 and FP2 after double thermal cycles had the worse deformation ability by tensile test. The toughness of FP2 improved after a single thermal cycle, decreased after double thermal cycles, and improved again after triple thermal cycles. The content of martensite–austenite (M–A) constituents and the average grain size of FP2 in the cascade samples were inversely proportional to FP2 toughness. Massive M–A constituents and their unique distribution at the inter-critical temperature were harmful to weld metal toughness. Controlling the size and fraction of M–A constituents can improve weld metal toughness.