AbstractStaphylococcus aureus is an important cause of morbidity and mortality in children in sub-Saharan Africa (SSA). A major risk factor for staphylococcal infection is S. aureus colonization of the anterior nares. We sought to define risk factors for S. aureus carriage and characterize antimicrobial resistance patterns in children in Botswana. A cross-sectional study was conducted at two clinical sites in southern Botswana. Patients under 18 years of age underwent two nasal swabs and brief interviews, 4 weeks apart. Standard microbiological techniques were used. For persistent carriers, S. aureus was isolated from swabs at both time points, and for intermittent carriers, S. aureus was isolated from only one swab. Poisson regression with robust variance estimator was used to compare prevalence of carriage and the resistance phenotypes. Among 56 enrollees, prevalence of S. aureus colonization was 55% (N = 31), of whom 42% (N = 13) were persistent carriers. Of human immunodeficiency virus-infected children, 64% (N = 9) were carriers. Risk factors for nasal carriage included a history of tuberculosis (prevalence ratio [PR] = 1.60; 95% confidence interval [CI] = 1.02, 2.51; P = 0.040) and closer proximity to health care (PR = 0.89; 95% CI = 0.80, 0.99; P = 0.048). Prior pneumonia was more common among persistent rather than intermittent carriers (PR = 2.64; 95% CI = 1.64, 4.23; P < 0.001). Methicillin-resistant S. aureus (MRSA) prevalence was 13%. Of isolates tested, 16% were resistant to three or more drugs (N = 7/44). In summary, children in southern Botswana are frequently colonized with S. aureus. Antibiotic resistance, especially MRSA, is also widespread. Antibiotic recommendations for treatment of staphylococcal infections in SSA should take cognizance of these resistance patterns.