DMV spokesman Mike Marando says the resignation letter submitted by Thomas Demartini late Wednesday did not allude to the incident.
Demartini had been on paid leave since October. That's when the DMV verified Amber Yust's claim that the clerk who processed her driver's license application sent her a letter at home calling her sex change "a very evil decision."
Yust has filed a claim against the state seeking damages.
Marando says Demartini received sensitivity training last year after refusing to assist another transgender customer.
Efforts by The Associated Press to reach Demartini for comment have been unsuccessful.