Functional markers, which are designed using polymorphic sites in gene sequences and which result in phenotypic variation, have high efficiency in marker-assisted selection (MAS). xa5 is a recessive gene that confers broad-spectrum resistance to bacterial blight, and it has been widely used in rice breeding projects in all tropical regions. In this study, two functional markers [Tetra primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR) and kompetitive allele specific PCR (KASP)] were developed based on the variation of two bases in the coding region of the xa5 allele. The specificity and applicability of the two functional markers were verified in a breeding segregating population and a rice micro-core germplasm resource population. We conclude that these two newly-developed functional markers can effectively determine the homozygosity and heterozygosity of the target gene under different laboratory conditions. Thus, they can be used for genotyping the xa5 gene in molecular breeding programmes, thereby improving breeding efficiency.