<b><i>Introduction:</i></b> Diabetic nephropathy (DN) and hypertensive nephrosclerosis (HN) represent the most common causes of chronic kidney disease (CKD) and many patients progress to ­end-stage renal disease. Patients are treated primarily through the management of cardiovas­cular risk factors and hypertension; however patients with HN have a more favorable outcome. A noninvasive clinical approach to separate these two entities, especially in hypertensive patients who also have diabetes, would allow for targeted treatment and more appropriate resource allocation to those patients at the highest risk of CKD progression. <b><i>Meth­ods:</i></b> In this preliminary study, high-spatial-resolution matrix-assisted laser desorption/ion­ization (MALDI) mass spectrometry imaging (MSI) was integrated with high-mass accuracy MALDI-FTICR-MS and nLC-ESI-MS/MS analysis in order to detect tissue proteins within kidney biopsies to discriminate cases of DN (<i>n</i> = 9) from cases of HN (<i>n</i> = 9). <b><i>Results:</i></b> Differences in the tryptic peptide profiles of the 2 groups could clearly be detected, with these becoming even more evident in the more severe histological classes, even if this was not evident with routine histology. In particular, 4 putative proteins were detected and had a higher signal intensity within regions of DN tissue with extensive sclerosis or fibrosis. Among these, 2 proteins (PGRMC1 and CO3) had a signal intensity that increased at the latter stages of the disease and may be associated with progression. <b><i>Discussion/Conclusion:</i></b> This preliminary study represents a valuable starting point for a future study employing a larger cohort of patients to develop sensitive and specific protein biomarkers that could reliably differentiate between diabetic and hypertensive causes of CKD to allow for improved diagnosis, fewer biopsy procedures, and refined treatment approaches for clinicians.