Diabetic kidney disease (DKD) has been pointed out as a prominent cause of chronic andend-stage renal disease (ESRD). There is a genetic predisposition to DKD, although clinically relevantloci are yet to be identified. We utilized a custom target next-generation sequencing 70-gene panelto screen a discovery cohort of 150 controls, DKD and DKD-ESRD patients. Relevant SNPs for thesusceptibility and clinical evolution of DKD were replicated in an independent validation cohortof 824 controls and patients. A network analysis aiming to assess the impact of variability alongspecific pathways was also conducted. Forty-eight SNPs displayed significantly different frequenciesin the study groups. Of these, 28 withp-values lower than 0.01 were selected for replication.MYH9rs710181 was inversely associated with the risk of DKD (OR = 0.52 (0.28–0.97),p= 0.033), whilstSOWAHBrs13140552 andCNDP1rs4891564 were not carried by cases or controls, respectively(p= 0.044 and 0.023). In addition, theRGMArs1969589 CC genotype was significantly correlatedwith lower albumin-to-creatinine ratios in the DKD patients (711.8±113.0 vs. 1375.9±474.1 mg/gfor TC/TT; mean difference = 823.5 (84.46–1563.0);p= 0.030). No biological pathway stood out asmore significantly affected by genetic variability. Our findings reveal new variants that could beuseful as biomarkers of DKD onset and/or evolution.