peer reviewed ; The aims of this study were to conduct a single-step genome-wide association to identify genomic regions associated with milk urea (MU) and to estimate genetic correlations between MU and milk yield (MY), milk composition (calcium content (CC), fat percentage (FP), protein percentage (PP), and casein percentage (CNP)), and cheese-making properties (CMP) (coagulation time (CT), curd firmness after 30 min from rennet addition (a30), and titratable acidity (TA)). The used data have been collected from 2015 to 2020 on 78,073 first-parity (485,218 test-day records), and 48,766 s-parity (284,942 test-day records) Holstein cows distributed in 671 herds in the Walloon Region of Belgium. Data of 565,533 single nucleotide polymorphisms (SNP), located on 29 Bos taurus autosomes (BTA) of 6,617 animals (1,712 males) were used. Random regression test-day models were used to estimate genetic parameters through the Bayesian Gibbs sampling method. The SNP solutions were estimated using a single-step genomic BLUP approach. The proportion of the total additive genetic variance explained by windows of 50 consecutive SNPs (with an average size of ∼216 Kb) was calculated, and the top-3 genomic regions explaining the largest rate of the genetic variance were considered promising regions and used to identify potential candidate genes. Mean (standard deviation) MU was 25.38 (8.02) mg/dl and 25.03 (8,06) mg/dl in the first and second lactation, respectively. Mean heritability estimates for daily MU were 0.21 and 0.23 for the first and second lactation, respectively. The genetic correlations estimated between MU and MY, milk composition, and CMP were quite low (ranged from -0.10 (CC) to 0.10 (TA) and -0.05 (CT) to 0.13 (TA) for the first and second lactations, respectively). The top-3 regions associated with MU were located from 80.61 to 80.74 Mb on BTA6, 103.26 to 103.41 Mb on BTA11, and 1.59 to 2.15 Mb on BTA14. Genes including KCNT1, MROH1, SHARPIN, TSSK5, CPSF1, HSF1, TONSL, DGAT1, SCX, and MAF1 were identified as ...