Abstract Background Non-caseating granulomas are a hallmark histopathological finding of Crohn’s disease (CD). Studies have suggested that the presence of granulomas may indicate a more aggressive CD phenotype associated with a complicated clinical course, including stricturing and/or penetrating disease, need for biologic therapy, and need for surgery. As such, identification of genetic associations of granulomatous CD (GCD) may help elucidate disease pathogenesis, which in turn may optimize treatments and guide novel therapeutics to combat CD complications. There is relatively sparse genetic information known about CD subtypes, especially GCD. The aim of this study was to determine the extent of genetic variation between pediatric CD patients with and without a pathognomonic sub-mucosal granuloma detected at the time of diagnosis. Methods Whole-exome next-generation sequencing (WES) was performed on peripheral blood derived DNA from patients with GCD and non-GCD (NGCD). PLINK analysis was used to identify single nucleotide polymorphisms (SNPs) that were overrepresented in comparisons between groups, and subgroup allele frequencies were also compared to publically available, large population-based genomic data in gnomAD. The potential deleteriousness of single nucleotide variants was determined by the CADD scoring tool. Results WES was completed for 17 patients with GCD and 19 with NGCD. There were no significant differences in baseline clinical characteristics, treatments, nor 1-year outcomes between the groups. Overlap analyses between PLINK- and gnomAD-generated SNPs revealed significant enrichment in those associated with HLA-DQA1, LILRA1, SAA2, PCDHB, HLA-B, NOD2, and IGH shared by GCD and NGCD groups. In the meantime, GCD-specific (top candidates linked with HLA-B and MUC4) and NGCD-specific (top candidates linked with HLA-B and ACOT9) SNPs were sparse. Conclusions We are the first to examine WES-based genetic variation between treatment-naïve pediatric CD patients purely separated by the presence of a sub-mucosal epithelioid granuloma. While there were very few SNPs consistently differentiating between GCD and NGCD patients in our cohort using two distinct methodologies, we were able to identify several novel SNPs that were significantly enriched in pediatric CD patients compared to the control human genome. Our findings will require subsequent confirmation in larger but similarly scrutinized cohorts of patients.