Few studies have examined parental variables in relation to GLB youth outcomes. This study aimed to establish the reliability and validity of observational codes that examined parental acceptance, emotional support, and ambivalence, and to examine the impact of these parental variables on GLB youth internalizing and externalizing problems, substance use problem severity, and GLB negative identity. Participants included 36 GLB parent-youth dyads at baseline, and out of those, 27 completed the two year follow-up. Results provided support for the reliability and validity of the observational codes. Parental ambivalence was positively correlated to youth-report of parental rejection, but was unrelated to parents' self-reports of rejection. Additionally, parental acceptance of sexual orientation at baseline was inversely related to GLB youth externalizing symptoms two years later, and parental emotional support was inversely related to GLB youth internalizing symptoms and substance use problem severity; however, there were no associations with GLB negative identity. Parental ambivalence was not related to longitudinal outcomes. The results of this study have implications for interventions targeting GLB youth and their parents.