To investigate the polymorphism of two groups of male sterile lines and their maintainer lines, we used amplified fragment length polymorphism markers. Of the 64 primer pairs, five reproducibly demonstrated polymorphism. Group I had 27 polymorphic bands, and Group II had 24 polymorphic bands. The maximum number of polymorphic bands was produced by line 2 (18 polymorphic bands) followed by line 7 (15 polymorphic bands). Of the five primer pairs, the highest number of polymorphic bands was produced by primer pair E1/M4, and the smallest number of polymorphic bands was produced by primer pair E1/M2. DNA fingerprinting, germplasm characterization, and cytogenetic research have especially been emphasized in studies of bread wheat; these methods are highly useful to select appropriate accessions for plant improvement and hybridization through molecular breeding approaches to evolve suitable safeguard strategies.