This study aimed to identify and analyze the effects of both isothermal heat treatment temperature and residence time on the formation of mesophase in coal tar pitch, especially with respect to its microstructural and crystalline evolution. The formation and growth of mesophase resulted in a decrease in d002 and an increase in Lc, and the degree of such variation was larger when the isothermal heat treatment temperature was higher. In isothermally heat-treated pitch, two distinct domains were observed: less developed crystalline carbon (LDCC) and more developed crystalline carbon (MDCC). When pitch was isothermally heat-treated at 375 °C for 20 h, d002 was 4.015 Å in the LDCC and 3.515 Å in the MDCC. Higher isothermal heat-treatment temperatures accelerated the formation, growth, and coalescence of mesophase. Indeed, in the pitch specimen isothermally heat-treated at 425 °C for 20 h, d002 was 3.809 Å in the LDCC and 3.471 Å in the MDCC. The evolution of mesophase was characterized by pronounced inflection points in d002 curves. It was found that the emergence of these inflection points coincided with pronounced changes in the microstructure of mesophase. This finding confirmed the relationship between inflection points in d002 and the microstructure of mesophase.