Abstract The ability to accurately select reproductively efficient replacement heifers is critical to sustainable beef production. Previous studies used metabolomic assessment of blood plasma and found significant differences in several amino acids in poor performing and top performing replacement heifers. The objective of this study was to identify differences in plasma amino acid profiles, at weaning, in heifers with different pregnancy outcomes following estrus synchronization and artificial insemination (AI). At weaning, blood samples were collected from a group of replacement heifers (n = 47) and processed for plasma extraction. The heifers then underwent a fixed-time AI program at approximately 14 months of age followed by pregnancy status evaluation via ultrasound. Amino acid profiles were compared between heifers with differing reproductive outcomes following first service AI. High performance liquid chromatography (HPLC) was utilized to generate amino acid profiles of the blood plasma collected at weaning. A total of twenty amino acid levels were analyzed for each heifer: histidine, asparagine, taurine, serine, glutamine, arginine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, cystine, lysine, tyrosine, methionine, valine, isoleucine, leucine, and phenylalanine. Of the twenty amino acids analyzed, two were significantly different between heifers that remained open compared to heifers that were pregnant following the AI. Histidine was significantly lower in heifers that remained open (n = 28, 40.89±1.56 µM/L, P < 0.05) compared to those that were pregnant following AI (n = 19, 46.59 ±1.937 µM/L, P < 0.05). Similarly, cystine was significantly lower in the heifers that remained open (n = 28, 7.87 ±2.05 µM/L, P < 0.05) compared to those that were pregnant (n = 19, 9.54 ±2.59 µM/L, P < 0.05). In conclusion, histidine and cystine plasma levels were found to be significantly different, in the blood plasma of heifers at weaning with differing AI first service reproductive outcomes.