To explore the mutagenic properties of the nucleotide analogue bromodeoxyuridine triphosphate (BrdUTP), the wild type α-amylase (xamy) gene from Xanthomonas campestris pv. campestris 8004 was used as a mutational target. It was mutated using PCR techniques to partially replace deoxythymidine triphosphate (dTTP) with BrdUTP. A total of 18 mutants were selected for DNA sequencing from the mutagenesis libraries by their ability to hydrolyze the starch. The results showed that 70% of the total mutations were single base-pair substitutions; BrdUTP also induced deletion and insertion mutation types. Among single base-pair substitutions, the predominant mutation type is transition (84%), but three kinds of transversions (16%) were also detected. It thus mainly induces A:T → G:C and T:A → C:G transitions. This result indicated that when bromouracil is present as a deoxyribonucleoside triphosphate substrate it mainly paired with dAMP, and when it is present as a template base it could pair with free dGTP. Three mutational hot spots induced by BrdUTP were revealed in this work.