EBT4 was newly released for radiotherapy quality assurance to improve the signal-to-noise ratio in radiochromic film dosimetry. It is important to know its dose-response characteristics before its use in the clinic. This study aims to investigate and compare the dose-response curves of the Gafchromic EBT4 film for megavoltage and kilovoltage x-ray beams with different dose levels, scanning spatial resolutions, and sizes of region of interest (ROI). EBT4 film (Lot#07052201) calibration strips (3.5×20cm2 ) were exposed to a 10×10cm2 open field at doses of 0, 63, 125, 500, 750, 1000cGy using 6MV photon beam. EBT4 film strips from the same lot were then exposed to each x-ray beam (6MV, 6MV FFF, 10MV FFF, 15MV, and 70kV) at six dose values (50, 100, 300, 600, 800, 1000cGy). A full sheet (25×20cm2 ) of EBT4 film was irradiated at each energy with 300cGy for profile comparison with the treatment planning calculation. At two different spatial resolutions of 72 and 300dpi, each film piece was scanned three consecutive times in the center of an Epson 10000XL flatbed scanner in 48-bit color. The scanned images were analyzed using FilmQA Pro. For each scanned image, an ROI of 2×2cm2 at the field center was selected to obtain the average pixel value with its standard deviation in the ROI. An additional ROI of 1cm diameter circle was also used to evaluate the impact of ROI shape and size, especially for FFF beams. The dose value, average dose-response value, and associated uncertainty were determined for each energy and relative responses were analyzed. The Student's t-test was performed to evaluate the statistical significance of the dose-response values with different color channels, ROI shapes, and spatial resolutions. The dose-response curves for the five x-ray energies were compared in three color channels. Weak energy dependence was found among the megavoltage beams. No significant differences (average ∼1.1%) were observed for all doses in this study among 6 MV, 6 MV FFF, 10 MV FFF, and 15 MV beams, regardless of spatial resolution and color channel. However, a statistically significant difference in dose-response was observed up to 12% between 70kV and 6MV beams. The dose-response curves for Gafchromic EBT4 films were nearly independent of the energy of the photon beams among 6MV, 6MV FFF, 10MV FFF, and 15MV. For very low-energy photons (e.g., 70kV), a separate calibration from the same low-energy x-ray is necessary.