Johne’s disease is a contagious bacterial infection in cattle caused by Mycobacterium avium ssp. paratuberculosis (Map) infection. A previous genome wide association study (GWAS) in Holstein cattle identified QTL on bovine chromosome 3 (BTA3) and BTA9 that were highly associated (P < 5 × 10⁻⁷) and on BTA1, BTA16, and BTA21 that were moderately associated (P < 5 × 10⁻⁵) with Map tissue infection. The objective of this study was to validate these GWAS results in Holsteins from the Pacific Northwest (PNW, n = 191) and a combined population from the PNW and the Northeast (PNW+NE, n = 432). DNA was genotyped using the Illumina BovineSNP50 BeadChip. Cases were ileo-cecal node positive for Map by PCR, and controls were Map tissue negative. Individuals were removed if the SNP call rate was < 90%, and SNPs were removed if the genotype call rate was < 95% or they had a MAF of < 0.01. After filtering, 162 cases, and 247 controls and 44,445 SNPs remained for analysis. A GWAS for the PNW and PNW+NE was conducted using an efficient mixed-model association eXpedited (EMMAX) method using 3 gene effect models. No loci were associated with the recessive model in PNW or PNW+NE. For the PNW cows, 4 dominant QTL were identified; 3 on BTA21 (P = 1.18 × 10⁻⁶, 8.15 × 10⁻⁶, 2.13 × 10⁻⁵) and 1 on BTA3 (9.88 × 10⁻⁶). In the additive model, a new QTL on BTA21 (5.14 × 10⁻⁷) and on BTA1 (P = 5.82 × 10⁻⁵) were identified. In the PNW+NE population, 2 QTL were identified with the dominant model: one on BTA14 (P < 3.17 × 10⁻⁵) and one on BTA16 (P < 3.17 × 10⁻⁵). The BTA16 QTL was also identified by the additive model (P < 4.59 × 10⁻⁵) and identified previously by our group using the NE cows alone (P < 2.57 × 10⁻⁵). SNPs associated with Map tissue infection on BTA16 lie within introns of CDC42BPA, a Serine/Threonine-protein kinase that has downstream effects on CDC42. Through the regulation of CDC42, CDC42BPA has been linked to several immunological pathways in humans including B-cell receptor and chemokine signaling pathways. Although none of the previous QTL in the NE GWAS were validated in the PNW population, a QTL on BTA16 was associated with susceptibility to Map tissue in both the NE and PNW+NE populations.