Analysis of flagellin genes was carried out on strains of Salmonella Typhimurium, Salmonella Hadar, Salmonella Abortusequi, Salmonella Enteritidis and Salmonella Gallinarum serovars, using a PCR system designed in this study. The purpose of these studies was to explore the flagellin genes of biphasic and monophasic Salmonellae for future targeted genetic interventions. The PCR primers were designed for two different structural genes of flagellin (fliC, fljB), for the repressor of fliC (fljA), for the operator region of fliC, and for the invertase system responsible for phase variation in Salmonella (hin, hixL, hixR). PCR analysis revealed that all of the examined genes (fliC, fliC-operator, fljB, fljA, hin, hixL, hixR) were present in all S. Typhimurium (n = 10) and S. Hadar (n = 10) strains tested. The results obtained on S. Typhimurium and S. Hadar strains confirmed their biphasic character at DNA level. However, the S. Enteritidis (n = 46) and S. Gallinarum (n = 5) strains lacked the invertase system (hin, hixL, hixR) as well as the fljA and fljB genes, while fliC and its operator were detectable. Consequently, the S. Enteritidis strains could only express fliC gene resulting in phase H1 flagellin. The examined S. Gallinarum strains were also demonstrated to have a cryptic flagellin gene (fliC). On the other hand, PCR results on S. Abortusequi (n = 2) indicated that both flagellin genes (fliC, fljB) and the whole phase variation system were present in both strains tested but only the H2 phase gene (fljB) was expressed. The phenotype of these strains could be clarified by motility test and/or by classical flagellar serology. The findings are also substantiated by the results of serovar-specific PCR for S. Typhimurium and S. Enteritidis. In conclusion, the PCR system developed in this study proved to be suitable for characterisation of Salmonella flagellin genes and confirmed serological results regarding all S. Typhimurium, S. Hadar and S. Enteritidis strains. This system could also identify cryptic flagellar genes of S. Abortusequi and S. Gallinarum.