Background Evolutionarily conserved sequences likely have biological function. Methods To determine whether variation in conserved sequences in non-coding DNA contributes to risk for human disease, we studied six conserved non-coding elements in the Th2 cytokine cluster on human chromosome 5q31 in a large Hutterite pedigree and in samples of outbred European American and African American asthma cases and controls. Results Among six conserved non-coding elements (>100 bp, >70% identity; human-mouse comparison), we identified one single nucleotide polymorphism (SNP) in each of two conserved elements and six SNPs in the flanking regions of three conserved elements. We genotyped our samples for four of these SNPs and an additional three SNPs each in the IL13 and IL4 genes. While there was only modest evidence for association with single SNPs in the Hutterite and European American samples (P < 0.05), there were highly significant associations in European Americans between asthma and haplotypes comprised of SNPs in the IL4 gene (P < 0.001), including a SNP in a conserved non-coding element. Furthermore, variation in the IL13 gene was strongly associated with total IgE (P = 0.00022) and allergic sensitization to mold allergens (P = 0.00076) in the Hutterites, and more modestly associated with sensitization to molds in the European Americans and African Americans (P < 0.01). Conclusion These results indicate that there is overall little variation in the conserved non-coding elements on 5q31, but variation in IL4 and IL13, including possibly one SNP in a conserved element, influence asthma and atopic phenotypes in diverse populations.