Sex differences exist in the structure and function of the cholinergic septo-hippocampal system throughout the lifespan of mammals. How and when these sex differences originate is unclear. Because estrogen modulates sexual differentiation of several brain regions during development and influences neurogenesis in adult mammals, we hypothesized that sexual dimorphism of the cholinergic septo-hippocampal system would extend to its neurogenesis. A birthdating agent 5'-bromo-2'-deoxyuridine (BrdU) was injected into pregnant dams on one of eight gestational days, ranging from embryonic day (E)10 to E17. The offspring were euthanized at 2 months of age, and brains were processed for BrdU and choline acetyltransferase (ChAT) immunoreactivity to label cholinergic neurons that became postmitotic on a given embryonic day and survived to adulthood. Unbiased stereology was used to compare the number of double-labeled neurons in the medial septum (MS) of female and male offspring. Cholinergic neurons in the MS were generated primarily between E11 and E14, similar to other published reports. We found sex differences in the pattern of peak neurogenesis but not in the length of neurogenesis, or in total number of neurons generated in the MS. Additionally, in adult female and male mice, we estimated the total number of cholinergic neurons using unbiased stereology and found no sex differences in the number of cholinergic neurons or in the volume of the MS in adulthood. These results suggest that sex differences noted in the function of the postnatal cholinergic septo-hippocampal system may originate from its neurogenesis.