Campylobacter is considered to be the leading bacterial etiologic agent of acute gastroenteritis in humans. Evidence implicates poultry as a major source of the organism for human illness; however, the pathways involved in Campylobacter contamination of poultry flocks, horizontal transmission and/or vertical transmission, remain unclear. Recent evidence implicates breeders as a potential source for Campylobacter contamination of the subsequent broiler offspring. In this investigation, Campylobacter isolated from feces, cloacal swabs, ceca, semen, and vas deferens of 12 breeder broiler roosters were genotyped by both flagellin A short variable region (flaA SVR) DNA sequence analysis and repetitive element (rep)–polymerase chain reaction (PCR). In 9 of 12 roosters, Camplylobacter was isolated from multiple sites sampled. Comparison of multiple isolates obtained from individual roosters revealed variable results. In five of the nine roosters, all Campylobacter isolated demonstrated closely related flaA SVR DNA sequences as well as rep-PCR patterns; isolates from these roosters were collected from both the gastrointestinal and the reproductive tracts or from the gastrointestinal tract alone. The remaining four roosters had Campylobacter that were distinct by both typing methods. Isolates from two of these four roosters originated from both the gastrointestinal and the reproductive tracts. Isolates from the remaining two roosters originated from only the reproductive tract. Comparisons of all Campylobacter isolates recovered from a distinct sample type within either the reproductive tract or the gastrointestinal tract (feces, semen, cloaca, vas deferens, or ceca) were quite diverse. No relationship between the genotypes and the sample type could be ascertained. Further investigation is needed to determine the route of contamination and if the presence of Campylobacter within the rooster leads to contamination of the broiler offspring via the fertilized egg.