Objective - To identify chromosomal regions associated with cranial cruciate ligament rupture (CCLR) in a population of Newfoundlands. Animals - 90 client-owned Newfoundlands. Procedures - A pedigree was constructed for dogs that did or did not have CCLR (determined on the basis of physical examination and radiographic findings). From this pedigree, affected and unaffected dogs were selected for genotyping on the basis of their predicted statistical likelihood of being homozygous CCLR-unaffected (n = 53) or homozygous CCLR-affected (37) dogs. Genotyping was performed for 532 microsatellite markers (MSATs). Comparisons of genotypes and allele frequencies were made between CCLR-affected and CCLR-unaffected dogs. Results - In the selected population, 495 MSATs were informative with a mean interval between markers of 5.5 centimorgans. Eighty-six MSATs were significantly associated with the CCLR trait, whereas 4 markers (located on 4 chromosomes) were significantly associated with the trait when false discovery rate (q value) was controlled at the 0.05 level. Subsequent initial validation confirmed significant trait association for 3 of the 4 MSATs. Conclusions and Clinical Relevance - In the population of Newfoundlands, 4 MSATs that were located on 4 chromosomes were significantly associated with the CCLR trait. Three of those markers were validated in part via genotyping additional closely located markers. The MSATs that were associated with the CCLR trait were identified in all regions (except for those on chromosome 24). Newfoundlands with CCLR could be used to study the disease process associated with anterior cruciate ligament injuries that occur in young female human athletes.