ABSTRACT Genotypic diversity in two flagellin genes, fliC and fljB, encoding phase-1 and phase-2 flagellin of Salmonella enterica, offers a potential biomarker for Salmonella subtyping. Forty-seven Salmonella isolates of 20 different serovars derived from chicken samples in Thailand were studied using the fliC/ fljB PCR-based RFLP assay. With two restriction endonucleases, MboI and HhaI, the fliC showed 11 and 9 patterns, while the fljB showed 6 and 7 patterns respectively. Though the PCR-based RFLP test cannot replace serotyping, the assay is based on the flagellin genes encoding proteins on the bacterial surface that are related to serotyping scheme. Overall, the assay was reproducible and successfully applied to simply screen Salmonella serovars as an alternative subtyping test for rapid traceability of Salmonella contamination in chicken production