Significance This study shows successful experimental phasing methods (single-wavelength anomalous diffraction, single isomorphous replacement, and single isomorphous replacement with anomalous scattering) for crystal structure determination of a membrane protein by serial femtosecond crystallography with X-ray free electron lasers. Our iodine-containing detergent provided strong anomalous and isomorphous difference signals, which enabled experimental phasing using lower-resolution reflections (worse than 3 Å) from fewer indexed images than phasing attempts reported previously. The findings of this study will be applicable to a wide range of target proteins in structural biology, especially membrane proteins that often diffract to low resolution.