Polysubstance use and the associated adverse consequences such as the ongoing and exponential increases in drug overdose deaths are major public health threats in the United States. There has been a substantial prevalence of driving under the influence of drugs (4.9% drivers in the United States) and drug-involved collisions (10.2% in Kentucky). In this study, 26 drugs including stimulants, opioids, and antipsychotics were measured in raw wastewater collected from two rest areas and a commercial truck service facility along two interstate highways in Kentucky. Methamphetamine, amphetamine, and cocaine were detected in all wastewater samples (n = 56) at both rest areas (I-24 E and I-24 W) and a commercial truck serving facility (I-75 N/S). Methamphetamine was discharged significantly higher at the rest areas (108 and 239 mg/d/1000 people at I-24 E and I-24 W, respectively); however, prescription opioids including tramadol, hydrocodone, and morphine as well as temazepam (benzodiazepine) and citalopram (antidepressant) were significantly discharged higher at the commercial truck service facility. Xylazine, a veterinary sedative, was also quantified for the first time in greater than 33% of collected wastewater samples. To the authors’ knowledge, this is the first quantitative study of drugs discharged at rest area facilities along interstate highways.