In order to understand and improve fracture toughness of heat affected zones (HAZs) of high-strength low alloy (HSLA) steels, complex microstructures including quasi-polygonal ferrite (QPF), acicular ferrite (AF), granular bainite (GB), bainitic ferrite (BF), and martensite-austenite (MA) constituent should be identified, quantified, and then correlated with critical crack tip opening displacement (CTOD). In this study, microscopic analysis methods were achieved for identification and quantitation of microstructures in the HAZs of three HSLA steels. The coarse-grained HAZ (CGHAZ) consisted of AF, GB, and BF together with a small amount of MA, while the inter-critically heated HAZ (ICHAZ) consisted of QPF, GB, and MA. In the CGHAZ, Ni promoted the formation of AF, while it prevented the formation of GB, and the addition of Ni resulted in very high critical CTOD. In the CGHAZ, both Ni and Mn promoted the formation of AF and prevented the formation of GB, while Ni was more effective than Mn. Thus, the addition of Ni resulted in very high critical CTOD. In the ICHAZ, both Ni and Mn promoted the formation MA. However, in the high-Ni-containing steel, a number of MAs were densified along Ni-segregated bands, and thus readily provided void initiation sites. This played an important role in reducing the mean free path for coalescence of voids and crack propagation, which easily led to the serious deterioration of critical CTOD.