Background The association between allergens in schools and childhood asthma has not been well studied, particularly in the United States. Objective To investigate allergen exposure in schools compared with homes with a specific focus on children with asthma. Methods Dust samples were collected from 46 rooms in 4 urban elementary schools (northeastern United States) and from 38 student bedrooms. Samples were analyzed for cat (Fel d 1), dog (Can f 1), cockroach (Bla g 2), dust mites (Der f 1/Der p 1), and mouse urinary protein (MUP). Questionnaires identified students with physician-diagnosed asthma. Results Cat and dog allergens were detectable in most school samples (96% and 78%, respectively), but at low levels. Cockroach allergen was detectable in only 11% of school samples. Mouse allergen was detectable in 89% of school samples, with 68% having MUP levels greater than 0.5 μg/g. In contrast, MUP was detectable in only 26% of bedroom samples. Matched classroom and home samples from 23 asthmatic students showed higher geometric mean MUP levels in the classroom vs the home (6.45 μg/g vs 0.44 μg/g, P < .001). However, there were lower geometric mean dust mite (Der f 1) levels in the classroom vs the home (0.04 μg/g vs 0.66 μg/g, P < .001). Conclusions There are significantly higher levels of MUP but lower levels of Der f 1 in schools vs homes. It is important to recognize that children with asthma may encounter varying levels of allergens in environments outside the home, such as schools.