Discovery of single nucleotide polymorphisms (SNPs) may lead to development of marker panels predictive of tolerance to high dietary nitrate (NO3-). The aims of this research were to identify SNPs in Argininosuccinate Lyase (ASL), determine the relationship of ASL SNP genotypes on NO3- tolerance, and determine relationships between ASL SNPs and genes previously identified as differentially expressed in response to elevated dietary NO3-. Liver tissues were collected from Suffolk ewes identified as highly tolerant (n = 6) or lowly tolerant (n = 6) to an 8 d administration of 300 mg KNO3/kg of BW 3 x daily. Tissues were also collected from untreated control (n = 6) ewes. Hot Star PCR and iPLEX Gold protocols were used for ASL SNP identification and genotyping, and the MIXED procedure of SAS was used to determine SNP relationships on NO3- tolerance and expression of previously identified genes. Nine polymorphic SNPs were identified within the ASL sequence. None of the ASL SNPs were related (P ≥ 0.468) to NO3- tolerance. However, eight ASL SNPs were correlated (P ≤ 0.048) with expression of SCP2, one SNP with FADS2, and two SNPs with KIK-I, genes all previously identified by microarray analysis as differentially expressed in response to high dietary NO3-. Tendencies (P ≤ 0.094) were also detected between ASL SNPs and expression of AOX1, ASL, and PFKFB1 genes. Overall, nine polymorphic SNPs within the ASL gene were linked to expression of genes identified as differentially expressed among ewes more and less tolerant to elevated dietary NO3-.