Adolescence is associated with continued maturation of the cerebral cortex, particularly the medial prefrontal cortex (mPFC). We have previously documented pruning in the number of neurons, dendrites and synapses in the rat mPFC from preadolescence to adulthood, with the period of pubertal onset being particularly important. We hypothesized that dopaminergic innervation of this region, critical for executive functions, would also be influenced by pubertal onset. Here, we measured changes in the volume of tyrosine hydroxylase (TH) immunoreactive axons in all layers of the male and female mPFC from preadolescence to adulthood (postnatal day (P) 25, 35, 45, 60 and 90) as a marker of dopaminergic innervation. Assessing both total fiber volume and length, TH fibers were quantified by multiplying the mPFC volume by fiber density. While there were subtle layer-specific changes, TH fiber volume and length increased between P25 and P90 in both males and females. Contrary to our hypothesis, a role for pubertal onset in TH innervation of this region was not discernable. In summary, axons immunoreactive for TH increase with similar trajectories in the mPFC of male and female rats from pre-puberty to young adulthood.