Despite the widespread recognition of pyuria in acute Kawasaki disease (KD) patients and its inclusion in the American Heart Association list of supporting laboratory data for KD diagnosis, no systematic study of pyuria and the origin of these cells in KD patients have been reported. We used automated urinalysis with flow cytometry to characterize urine samples from 135 acute KD subjects and 87 febrile control (FC) subjects without urinary tract infection. Pyuria [defined as ≥12 (for males) or 20 (for females) cells/μL] was present in 79.8% of KD and 54.0% of FC subjects (P < 0.0001). The median number of white blood cells (WBC) in the urine was 42 WBC/μL in KD and 12 WBC/μL in FC (P < 0.0001). No significant difference between the groups was seen for urine red blood cell (RBC) count, protein, or specific gravity. Comparison of voided versus catheter-collected urine samples indicated an origin of the cells from the bladder or upper urinary tract in both patient groups. Pyuria in KD subjects was not correlated with age or day of illness. Overall, the presence of pyuria was neither specific nor sensitive as a marker for KD, but the magnitude of pyuria was significantly higher in KD patients compared with the FC group.