In this work, we investigate the influence of defect concentration of the diamond substrates on the performance of hydrogen-terminated diamond field-effect transistors by Raman spectra, pulsed I–V characteristics analysis, and radio frequency performances measurements. It is found that a sample with higher defect concentration shows larger drain-lag effect and lower large-signal output power density. Defects in the diamond act as traps in the carrier transport and have a considerable influence on the large-signal output power density of diamond field-effect transistors. This work should be helpful for further performance improvement of the microwave power diamond devices.