Puberty is a fundamental developmental process experienced by all reproductively competent adults, yet the specific factors that regulate variation in its timing remain elusive. Using a new approach to identifying these factors, we have performed a survey among a panel of chromosome substitution strains (for inbred strains C57BL/6J and A/J) followed by linkage analysis to map a quantitative trait locus (QTL) on the distal end of chromosome 6 that regulates pubertal timing (as assessed by vaginal opening) in mice. The location of the QTL was then refined to a region between marker D6MIT59 and the end of the chromosome by generating and phenotyping a panel of 12 congenic strains, each with a unique and overlapping homozygous segment of the A/J chromosome on an otherwise uniform C57BL/6J genomic background. Additional characterization of the QTL indicated that the effects of the responsible gene(s) are gender specific and inherited in a codominant manner without parent-of-origin effects. These findings represent an important advancement toward identification of novel factors that regulate maturation of the hypothalamic-pituitary-gonadal axis and determine the timing of puberty.