Background: Specific biomarkers of pyelonephritis (PN) in cats are lacking. MicroRNAs (miRNAs) have diagnostic potential in human nephropathies. Objectives: To investigate the presence/stability of miRNAs in whole urine of cats and the discriminatory potential of selected urinary miRNAs for PN in cats. Animals: Twelve healthy cats, 5 cats with PN, and 13 cats with chronic kidney disease (n = 5), subclinical bacteriuria (n = 3), and ureteral obstructions (n = 5) recruited from 2 companion animal hospitals. Methods: Prospective case-control study. Expression profiles of 24 miRNAs were performed by quantitative PCR (qPCR). Effect of storage temperature (4°C [24 hours], −20°C, and −80°C) was determined for a subset of miRNAs in healthy cats. Results: Urinary miR-4286, miR-30c, miR-204, miR4454, miR-21, miR-16, miR-191, and miR-30a were detected. For the majority of miRNAs tested, storage at 4°C and −20°C resulted in significantly lower miRNA yield compared to storage at −80°C (mean log2fold changes across miRNAs from −0.5 ± 0.4 SD to −1.20 ± 0.4 SD (4°C versus −80°C) and from −0.7 ± 0.2 SD to −1.20 ± 0.3 SD (−20°C versus −80°C)). Cats with PN had significantly upregulated miR-16 with a mean log2fold change of 1.0 ± 0.4 SD, compared with controls (−0.1 ± 0.2, P =.01) and other urological conditions (0.6 ± 0.3, P =.04). Conclusions: Upregulation of miR16 might be PN-specific, pathogen-specific (Escherichia coli), or both.