The small heat shock protein (HSP) B family of proteins are a group of molecular chaperones that enable tissues to adapt to changes in their local environments during differentiation, stress, or disease conditions. The objective of this research was to characterize the expression of HSPB8 and its cochaperone Bcl2-associated athanogene 3 (BAG3) in nonpregnant (NP) and pregnant rat myometrium during myometrial programming. Rat myometrium was collected from NP and pregnant rats as well as 1 day postpartum (PP) and samples prepared for immunoblot and immunofluorescence analysis. Immunoblot analysis determined that HSPB8 protein expression was significantly elevated at Day (D) 15, D17, and D19 compared to expression at NP and D6, while BAG3 expression was significantly elevated at D15 compared to NP, and D17 compared to NP, D6, D23, and PP time points (P < 0.05). In situ, HSPB8 and BAG3 were predominantly localized to myometrial cells throughout pregnancy, with intense cytoplasmic HSPB8 and BAG3 detection on D15 and D17 in both longitudinal and circular muscle layers. Immunoblot analysis of HSPB8 and BAG3 protein expression in myometrium from unilateral pregnancies also revealed that expression of both proteins was significantly increased at D15 in gravid compared to nongravid horns. Thus, HSPB8 and BAG3 are highly expressed during the synthetic phase of myometrial differentiation marked by initiation of uterine distension and myometrial hypertrophy. HSPB8 and BAG3 could be regulators of the protein quality control required for this process.