Background. On the basis of a polymerase chain reaction-restriction fragment-length polymorphism analysis of the 16S-23S ribosomal DNA intergenic spacer, clinical isolates of Borrelia burgdorferi can be classified into 3 genotypes designated as RST1, RST2, and RST3. RST1 strains are the most pathogenic, and RST3 strains are the least pathogenic. Methods. Human leukocyte antigen (HLA) class II alleles were determined for a group of culture-positive patients with Lyme disease-associated erythema migrans and were evaluated for an association with the genotype of the infecting B. burgdorferi strain. Results. The DRB1*0101 allele carriage rate was higher in patients infected with RST3 strains (9/25 [36.0%]) than in patients infected with RST1 strains (2/28 [7.1%]) or RST2 strains (7/36 [19.4%]) (P = .010). The same relationship was found for carriage of the DRB1*0101-DQB1*0501 haplotype (P = .018), because of tight linkage disequilibrium. Similar associations could not be demonstrated for any of the other DRB1 and DQB1 alleles or haplotypes that were assessed. Conclusion, The DRB1*0101 allele and the DRB1*0101-DQB1 *0501 haplotype may be relevant to the development of infection with strains from the least invasive genotypes of B. burgdorferi. © 2005 by the Infectious Diseases Society of America. All rights reserved.