Previous studies evaluating single and multiple GnRH injections at time of CIDR removal have reported decreased interval to estrus and increased expression of estrus, and increased conception rates, respectively. Thus, the objective of this study was to compare the effects of supplementing GnRH at CIDR removal on expression of estrus and conception rates in beef heifers following the 7-d CO-Synch + CIDR protocol. Heifers (n = 1,482) from 11 herds over two years were synchronized using the 7-d CO-Synch + CIDR fixed time AI (FTAI) protocol and were randomly assigned to receive one of three treatments at CIDR removal [0 µg GnRH (0); 5 µg GnRH (5); or 5 µg at CIDR removal and 5 µg 12 h later (5 + 5)]. Expression of estrus and conception rates were analyzed using the GLIMMIX procedure of SAS with treatment as an independent variable and herd as a random effect. There was no effect of treatment on expression of estrus (P = 0.38), or AI conception rates (P = 0.85). There was an effect of estrus (P < 0.01) and a treatment × estrus interaction (P = 0.02). Animals that exhibited estrus prior to FTAI had increased (P < 0.01) conception rates compared with those that did not. Animals that exhibited estrus had increased (P < 0.01) conception rates compared with non-estrus animals in the 0 and 5 + 5 µg groups (54 ± 3% vs 34 ± 3%, and 52 ± 3% vs 36 ± 3%, respectively), but in the 5 µg group, there was no difference (P = 0.53) in conception rates between estrus and non-estrus animals (44 ± 3% vs 41 ± 3%, respectively). In conclusion, estrus expression increased pregnancy success, but supplementation with 5 or 5 + 5 µg of GnRH at CIDR removal did not improve estrus response or conception rates in beef heifers.