It is essential to inspect manufactured and in-service machines and components in industry. Many different are currently in use for this purpose. Ultrasonic testing is one of the most important nondestructive testing methods. Ultrasonic testing has different applications such as defect detection, assessment of mechanical and metallurgical properties of materials and temperature measurement. In the first part of this paper, theoretical equations and finite element analysis of variation of longitudinal ultrasonic wave velocity in the presence of a thermal gradient were studied. In the second part, the effect of a thermal gradient on the longitudinal ultrasonic waves is investigated by experiments. A specific test rig is designed and fabricated for this purpose. It can provide the desired temperatures and transmit and receive ultrasonic bulk waves simultaneously. Twelve different tests were carried out to study the effects of the work piece length and maximum and minimum temperatures. The experimental results are compared with the theory under similar conditions and very good agreement is observed. Uncertainty analysis is incorporated for determining the uncertainty in measuring the ultrasonic wave velocity in the presence of a thermal gradient and identifying the sources of error. The measurements were found to be quite accurate with an uncertainty of 4.5 m/s.