Diagnosis of pyelonephritis in cats is usually based on clinical signs, laboratory testing, and ultrasonographic findings, although definitive diagnosis requires positive urine culture obtained via pyelocentesis. MicroRNAs have been studied as biomarkers of renal injury in humans. This prospective case-control study sought to evaluate the presence and stability of microRNAs in cat urine and the discriminatory potential of selected urinary microRNAs for pyelonephritis. Several microRNAs were detected in urine, although storage temperature affected yield. There was upregulation of miR-16 in cats with pyelonephritis, but further research is needed to determine whether this is pyelonephritis-specific, pathogen-specific (ie, Escherichia coli), or both.