Using data from a regional census survey of high school students, we compared substance use patterns between transgender and cisgender students, and students who did not know if they are transgender (DKT). Data from more than 23,000 youth from 26 high schools were used to compare alcohol-related behaviors, marijuana use, and non-medical use of prescription drugs (NMUPD) between these three groups. We also examined differences in substance use behaviors between these groups by grade. Results show that transgender students have significantly higher odds of engaging in most types of substance misuse behaviors including problem drinking, current marijuana use, and NMUPD, compared to cisgender students. DKT students have significantly higher odds of engaging in problem drinking, and NMUPD compared to cisgender students. Furthermore, differences in some substance use behaviors between transgender and cisgender students are greater among students in earlier grades compared to later grades; though this was not the case when comparing cisgender students to DKT students. Results highlight the elevated risk of substance use among transgender youth--especially during early high school years, and students who are not sure about their gender identity. Implications about the need for targeted and early prevention efforts for this vulnerable population are discussed.