Prenatal stress in beef cattle has the potential to result in unexpected, long term phenotypic changes to an animal’s performance. One possible explanation for this phenomenon is stress induced epigenetic changes to the genome (e.g., methylation or histone modifications). The objective of this experiment was to identify differential DNA methylation patterns in prenatally stressed (PNS) Brahman females. Multiparous Brahman cows (n = 48) were transported for a 2hr periods on 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 d of gestation. A non-transported group (n = 48) was maintained as a control. DNA was extracted from the white blood cells of their 28-d old heifer calves (Control n = 8, PNS n = 6) and methylation was assessed using reduced representation sodium bisulfite sequencing. Mean methylation ratios at locations across the genome in PNS and Control females were compared using a student’s t-test. Analysis revealed 16,377 CpG sites, 311 CHG, and 612 CHH (C= cytosine; G= guanine; H = adenine, cytosine or thymine) sites that were differentially methylated in PNS females compared to the Control females (P ≤ 0.05). The top 2,000 differentially methylated regions (DMRs) were classified by functional location in introns, exons, CpG islands, promoters, and 5’UTR regions. Methylation differences in these regions are known to cause gene expression differences. The identified regions were analyzed using Ingenuity Pathway Analysis (IPA) in order to identify involvement in canonical pathways. Certain regions identified from the hypermethylated DMRs showed the strongest overlap (P = 1.93E-6) with the complex pathway of axonal guidance signaling (important for neural development). These results suggest that prenatal transportation stress does alter the epigenome of Brahman heifers and can influence biological pathways, potentially impacting animal performance.