In a commercial broiler flock during rearing multiple genotypes of Campylobacter jejuni may be present as well as in gastrointestinal tracts of individual birds. The aim of this study was to optimize and apply a denaturing gradient gel electrophoresis assay of the flagellin gene (fla-DGGE) for analysis of C. jejuni and Campylobacter coli in cecal samples of broilers without prior cultivation. One C. coli and 21 C. jejuni strains isolated from broiler flocks, of which 14 typed as unique by restriction fragment length polymorphism of flaA and two undefined strains, were clustered into 9 groups when applying fla-DGGE. Spiking of cecal samples revealed that fla-DGGE is able to detect at least 4.55-5.96logCFUCampylobacter/mlcecal material. The presence of 3 strains spiked in cecal material was demonstrated by fla-DGGE as the corresponding bands were visible on the DGGE gel. Naturally contaminated cecal samples were shown to contain different types of C. jejuni and C. coli. Fla-DGGE has some potential as a cultivation-independent fast primary subtyping method for C. jejuni and C. coli in cecal samples of broilers.