Polysubstance use (PSU) is the use of more than one psychoactive substance simultaneously or independently, and occurs in roughly half of individuals who seek treatment for substance use. The current study sought to use resting state functional connectivity (rs-FC) to examine functional connectivity in participants who report using multiple or single substances. Participants were drawn from a larger neuroimaging study. From there, participants were placed into one of three groups based on their frequency of alcohol, tobacco, cannabis, and illicit drug use. The final sample consisted of 82 participants. We observed three clusters that differed significantly between the three groups; one within the salience network and two within the temporal network. Tri+ users were found to have a lesser amount of rs-FC in these regions (compared to the other two groups) and dual users were found to have a greater amount of rs-FC within these regions. Findings indicate that use of three or more substances may significantly impact rs-FC within the salience and temporal networks, and that those who use alcohol+cannabis have significantly greater rs-FC than those who use alcohol+tobacco. Research is needed to examine larger samples of PSU for comparisons across specific substance combinations.