Based on combined methods, this study investigated substance use and HIV risk behaviors among kathoey sex workers (KSWs) in Bangkok, Thailand. The study found that only half of the KSW participants reported having been tested for HIV, and that except for one participant, all others had not seen health care providers in the past 12 months. About one third of the participants reported having engaged in unprotected anal sex with customers in the past six months. Almost all participants reported alcohol use, as well as having had sex with customers under the influence of alcohol. The prevalence of marijuana and ecstasy use in the past 12 months was high (32 and 36%, respectively); as was for ketamine (20%) and non-injecting methamphetamine (yaba) use (10%). A multiple regression analysis showed that the participants who were post-operative status, had used illicit drugs, or had been abused by their father and brothers were less likely to use condoms for anal sex with customers. Three quarters of the participants sent money to their families and 35% of the participants expressed their willingness to engage in unsafe sex when customers offer extra money. The qualitative interviews revealed that many identified as girl or kathoey in early age and had been exposed to transphobia and violence from father and brothers. Some reported support for gender transition from their mothers. More than half of the participants currently had difficulties in living as kathoey, such as challenges in the job market and relationship with family members. Family obligation for sending money and the Buddhist concept of karma were discussed in relation to risk behaviors among KSWs. The study provided implications for facilitating HIV testing and developing future HIV prevention intervention programs for KSWs in Thailand.