Purpose: The purpose of this study was to investigate how speech naturalness relates to masculinity-femininity and gender identification (accuracy and reaction time) for cisgender male and female speakers as well as transmasculine and transfeminine speakers. Method: Stimuli included spontaneous speech samples from 20 speakers who are transgender (10 transmasculine and 10 transfeminine) and 20 speakers who are cisgender (10 male and 10 female). Fifty-two listeners completed three tasks: a twoalternative forced-choice gender identification task, a speech naturalness rating task, and a masculinity/femininity rating task. Results: Transfeminine and transmasculine speakers were rated as significantly less natural sounding than cisgender speakers. Speakers rated as less natural took longer to identify and were identified less accurately in the gender identification task; furthermore, they were rated as less prototypically masculine/feminine. Conclusions: Perceptual speech naturalness for both transfeminine and transmasculine speakers is strongly associated with gender cues in spontaneous speech. Training to align a speaker's voice with their gender identity may concurrently improve perceptual speech naturalness.