BACKGROUND: Mobile health (mHealth) interventions, including smartphone apps, have been found to be an effective means of increasing the uptake of HIV prevention tools, including HIV and sexually transmitted infection (STI) tests and pre-exposure prophylaxis. However, most HIV prevention mHealth apps tested in the United States have been tested among populations living in areas surrounding urban centers. Owing to reduced access to broadband internet and reliable cellular data services, it remains unclear how accessible and effective these interventions will be in rural areas. In addition, gay and bisexual men who have sex with men and gender minority populations in rural areas experience enhanced stigma when compared with their more urban counterparts, and these experiences might affect their willingness and interest in mHealth apps. OBJECTIVE: This study aimed to conduct online focus groups with men who have sex with men and transgender and gender diverse populations in the rural southern United States to assess their interest in mHealth HIV prevention apps and the features that they would be the most interested in using. METHODS: Focus group participants were recruited from a larger pool of sexual and gender minority respondents to a web-based research survey. The participants indicated that they would be willing to participate in an online focus group discussion. Focus groups were conducted via secure Zoom (Zoom Video Communications Inc) videoconferencing. During the focus group discussions, participants were asked to discuss their experiences with HIV and STI prevention and how these experiences were affected by living in a rural area. They were then shown screenshots of a new app to promote HIV and STI prevention among rural populations and asked to provide their opinions on the app's features. The transcripts of the discussions were reviewed and coded using a constant comparative approach. RESULTS: A total of 6 focus groups were conducted with 26 participants. Most participants were cisgender gay and bisexual men who have sex with men (19/26, 73%); the remaining participants were transgender men (2/26, 8%), were nonbinary people (2/26, 8%), or had multiple gender identities (3/26, 12%). Participants reported numerous barriers to accessing HIV and STI prevention services and accurate information about HIV and STI prevention options. Overall, the participants reported a high degree of interest in mHealth interventions for HIV and STI prevention and suggested several recommendations for the features of an app-based intervention that would be the most useful for rural residents. CONCLUSIONS: These focus group discussions indicate that rural residence is not a major barrier to mHealth HIV and STI prevention intervention implementation and that there is a high degree of interest in these approaches to HIV and STI prevention.