Introduction: Fine wool production is an important source of revenue, accounting for up to 13% of total revenue in extensively managed wool sheep production systems of the United States. The Rambouillet are a predominant breed that excels in wool quality characteristics. Understanding the genetic basis of wool quality characteristics would aid in the development of genomic breeding strategies to facilitate genetic improvement.Methods: Wool characteristics and DNA were collected for rams enrolled in the North Dakota State University and University of Wyoming annual central performance ram tests over a three-year period (2019–2021, N = 313). The relationships of wool quality characteristics including grease fleece weight adjusted 365 days (wt. 365 adj.), clean fleece wt. 365 adj., staple length 365 adj., average fiber diameter, face wool cover, amount of skin wrinkles and belly wool were evaluated through genome-wide association studies (GWAS), Pearson correlation and ANOVA.Results: The GWAS identified four genome-wide significant genetic markers (p-value <1.19e-06) and five chromosome-wide significant markers (p-value <1.13e-05) on chromosomes 1, 2, 4, 15, and 19. Significant markers were associated with genes notable for relevant wool biological functions, including the gene ABCC8 which codes for SUR1, an ATP-sensitive potassium channel known to affect hair growth and 60S ribosomal protein L17-like, previously found to be expressed during follicle formation. The strongest Pearson correlation coefficients were identified between clean fleece wt. 365 adj. and grease fleece wt. 365 adj. (r = 0.83) and between clean fleece wt. 365 adj. and staple length 365 adj. (r = 0.53). Additionally, clean fleece wt. 365 adj. was correlated with final body weight (r = 0.35) and scrotal circumference (r = 0.16). Staple length 365 adj. (p-value = 5e-04), average fiber diameter (p-value = .0053) and clean fleece wt. 365 adj. (p-value = .014) were significantly associated with belly wool score.Discussion: The results of this study provide important insight into the relationships between wool quality characteristics and report specific markers that Rambouillet sheep producers may use to help inform selection and breeding decisions for improved wool quality.