Heterogeneous stock (HS) animals provide the ability to map quantitative trait loci at high resolution [<5 Megabase (Mb)] in a relatively short time period. In the current study, we hypothesized that the HS rat colony would be useful for fine-mapping a region on rat chromosome 1 that has previously been implicated in glucose regulation. We administered a glucose tolerance test to 515 HS rats and genotyped these animals with 69 microsatellite markers, spaced an average distance of <1 Mb apart, on a 67 Mb region of rat chromosome 1. Using regression modeling of inferred haplotypes based on a hidden Markov model reconstruction and mixed model analysis in which we accounted for the complex family structure of the HS, we identified one sharp peak within this region. Using positional bootstrapping, we determined the most likely location of this locus is from 205.04 to 207.48 Mb. This work demonstrates the utility of HS rats for fine-mapping complex traits and emphasizes the importance of taking into account family structure when using highly recombinant populations.