Tsetse fly (Diptera: Glossinidae) viviparous reproductive physiology remains to be explored at the molecular level. Adult females carry their young in utero for the duration of embryonic and larval development, all the while supplying their offspring with nutrients in the form of a "milk" substance secreted from a modified accessory gland. Flies give birth to fully developed third instar larvae that pupariate shortly after birth. Here, we describe the spatial and temporal expression dynamics of two reproduction-associated genes and their products synthesized during the first and second gonotrophic cycles. The proteins studied include a putative yolk protein, Glossina morsitans morsitans yolk protein 1 (GmmYP1) and the major protein found in tsetse "milk" secretions (Glossina morsitans morsitans milk gland protein, GmmMGP). Developmental stage and tissue-specific expression of GmmYP1 show its presence exclusively in the reproductive tract of the fly during oogenesis, suggesting that GmmYP1 acts as a vitellogenic protein. Transcripts for GmmMGP are present only in the milk gland tissue and increase in coordination with the process of larvigenesis. Similarly, GmmMGP can be detected at the onset of larvigenesis in the milk gland, and is present during the full duration of pregnancy. Expression of GmmMGP is restricted to the adult stage and is not detected in the immature developmental stages. These phenomena indicate that the protein is transferred from mother to larvae as nourishment during its development. These results demonstrate that both GmmYP1 and GmmMGP are involved in tsetse reproductive biology, the former associated with the process of oogenesis and the latter with larvigenesis.