Background: Knee osteoarthritis (OA) is a single most arthritic disease. Knee joint space width (JSW) is commonly used for grading severity of knee OA. However, previous studies did not established criterion validity and test-retest reliability of ultrasound (US) image for measuring JSW. Objects: The aim of this study was to establish criterion validity and test-retest reliability of US measurement of medial and lateral knee JSW. Methods: Twenty-nine subjects with knee OA were participated. The US and X-ray were used to measure knee JSW. One sample Kolmogorov-Smirnov test was used to confirm the data normal distribution. Pearson correlation coefficient and ICC were used to calculated and establish criterion validity and test-retest reliability, respectively. Results: US measurement of medial and lateral knee JSW was highly correlated with radiographic imaging measure (r=.714 and .704, respectively). Test-retest reliabilities of medial and lateral knee JSW were excellent correlated (ICC=.959 for medial side and .988 for lateral side, respectively). Conclusion: US may be valid tool to measure knee JSW.