Many reports described the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in different livestock animals from one-species farms. However, in no published reports the prevalence on mixed poultry-pig farms was mentioned, nor the possible relation in MRSA colonization between those two species on one farm, and the possible role of the farmer in the dissemination of MRSA between those two species. Furthermore, no data is available on the optimal sampling site to detect MRSA in broilers. Therefore this study aimed to determine the most suitable sample location in broiler chickens for MRSA and the within flock prevalence of MRSA in various broiler flocks and compared this with the MRSA prevalence in pigs, the colonization of the farmer and the contamination in the barn environment in three mixed poultry-pig farms. MRSA was most frequently isolated from the cloaca and nose shell and to a lesser extent from the skin beneath the wing and the pharynx. The relative sensitivity of the different anatomical sites was, 44.4% for the cloaca, 33.3% for the nose shell, 16.7% for the skin beneath the wing and 5.6% for the pharynx. Based upon these relative sensitivities combining cloaca and nose shell would increase the chance of MRSA detection. A rather low within flock prevalence of MRSA varying between 0% and 28% was detected in broilers, whereas in pigs on the same farms the within herd prevalence varied between 82% and 92%. No MRSA contamination in the direct barn environment of the broilers was found, this in contrast to the environment of the pigs, indicating a relationship between MRSA prevalence and contamination in the environment. Two farmers were continuously colonized, while the third one was only once. In conclusion, a major difference was seen in MRSA occurrence between broilers and pigs from the same farm. This may suggest that broilers are naturally less susceptible to MRSA ST398 colonization than pigs. Conversely, short production time in broilers, vacancy of the barn environment during one week and the higher frequency of disinfection might also explain the lower prevalence in broilers. The farmer may play an important role in the dissemination of MRSA from pigs to poultry, especially in mixed farms where pigs are highly colonized and may act as a reservoir for MRSA ST398 carriage in humans.