The specific purpose was to investigate the possible interrelationships of genotypes of Staphylococcus aureus found in mammary glands, horn flies, and extramammary sites on 3 southeastern US dairies. A total of 1,228 samples were obtained from various sources on the 3 dairy herds, each of which had a history of Staph. aureus mastitis. Dairy herds studied had access to pasture, and samples were collected during the summer when horn flies (Haematobia irritans) were active. Samples collected included milk samples from all lactating herd cows, colostrum samples from heifers calving during the study period, heifer body sites (mouth, nostrils, and teats), the heifer environment (water, feed, and soil/vegetation/pasture), horn flies, and humans (hands and nostrils). Isolation of Staph. aureus was attempted from all samples, with isolates subjected to genotypic analysis using pulsed-field gel electrophoresis. A total of 244/1228 (or 19.9%) of all samples were positive for Staph. aureus. For milk samples, 52/383 (or 13.6%) of samples were Staph. aureus positive, and 70/411 (or 17.0%) of heifer quarter colostrum samples were positive. Horn fly samples were frequently positive, with over one-half (29/52, or 55.8%) of samples positive for Staph. aureus. Staphylococcus aureus obtained during the study comprised isolates from 12 different genotype groups as defined in this study. Identical genotypes were obtained from horn flies, heifer colostrum samples, and cow milk samples. Group B genotypes were shared among flies, heifer colostrum samples, body sites, and cow milk samples, whereas group A genotypes were common to the same sample locations and body sites but rarely (once) found in horn flies. We conclude, based upon the finding of identical pulsed-field gel electrophoresis genotypes in flies, heifer body sites, and heifer colostrum samples, that flies and heifer body sites could be important sources of Staph. aureus for heifer intramammary infections.