The objective of this study was to observe the effect of Pregnant Mare Serum Gonadotrophin (PMSG) administration time on progesterone (P4) and estradiol (E2) concentrations in synchronized ewes. The experimental animals used in this study were Cameroon hair sheep and Thai Long Tail wool sheep crossbreds. Controlled Internal Drug Release (CIDR) device was implanted intravaginally to 40 ewes for 11 and 13 days. Pregnant Mare Serum Gonadotrophin (PMSG), 200 I.U. was administered at CIDR withdrawal and 24 hours prior to CIDR withdrawal. Blood samples were collected between 3 to 4 day intervals for 6 weeks after CIDR implantation. The P4 and E2 blood plasma concentrations were measured by radioimmunoassay (RIA). Ewes that received PMSG at CIDR withdrawal gave significantly higher P4 and E2 concentrations as compared to ewes that received PMSG at 24 hours prior to CIDR withdrawal in both 11 and 13 days CIDR implantation. In conclusion, PMSG administration at 24 hour prior to or at CIDR withdrawal would affect the concentrations of blood plasma P4 and E2 in synchronized ewes and subsequently would affect the reproductive performance in ewes.