Recent studies have shown that livestock can carry Staphylococcus aureus and transmit it to human caretakers. We conducted a pilot study to determine the prevalence and molecular epidemiology of S. aureus among rural Iowans, including individuals with livestock contact. Nasal and throat swabs were collected and plated onto selective media to isolate methicillin-susceptible and methicillin-resistant S. aureus (MRSA), followed by antibiotic resistance testing and molecular analysis of the isolates. While no MRSA was detected, overall, 23.7% (31/131) of participants were found to harbor S. aureus in their nose, throat, or both. Fifteen isolates displayed resistance to one or more tested antibiotics, and the Panton-Valentine leukocidin (PVL) genes were present at a high level (29% [9/31] of S. aureus-positive participants). Younger age and tobacco use were associated with increased risk of S. aureus carriage. Our results suggest that carriage of PVL-positive S. aureus is common among rural Iowans, even in the absence of detectable MRSA colonization.