Meiotic recombination in flow-sorted single sperm was used to analyze four highly polymorphic microsatellite markers on the long arm of chromosome 9. The microsatellites comprised three tightly linked markers: 9CMP1 (D9S109), 9CMP2 (D9S127), and D9S53, which map to 9q31, and a reference marker, ASS, which is located in 9q34.1. Haplotypes of single sperm were assessed by using PCR in a single-step multiplex reaction to amplify each locus. Recombinant haplotypes were identified by their relative infrequency and were analyzed using THREELOC, a maximum-likelihood-analysis program, and an adaptation of CRI-MAP. The most likely order of these markers was cen-D9S109-D9S127-D9S53-ASS-tel with D9S109, D9S127, and D9S53 being separated by a genetic distance of approximately 3%. The order of the latter three markers did not however achieve statistical significance using the THREELOC program.