Remote-controlled (RC) unmanned aerial vehicles (UAVs) have been used to study the movement of agricultural threat agents (e.g., plant and animal pathogens, invasive weeds, and exotic insects) above crop fields, but these RC UAVs are operated entirely by a ground-based pilot and often demonstrate large fluctuations in sampling height, sampling pattern, and sampling speed. In this paper, we describe the development and application of an autonomous UAV for precise aerobiological sampling tens to hundreds of meters above agricultural fields. We equipped a Senior Telemaster UAV with four aerobiological sampling devices and a MicroPilot-based autonomous system, and we conducted 25 sampling flights for potential agricultural threat agents at Virginia Tech's Kentland Farm. To determine the most appropriate sampling path for aerobiological sampling above crop fields with an autonomous UAV, we explored five different sampling patterns, including multiple global positioning system (GPS) waypoints plotted over a variety of spatial scales. An orbital sampling pattern around a single GPS waypoint exhibited high positional accuracy and produced altitude standard deviations ranging from 1.6 to 2.8 m. Autonomous UAVs have the potential to extend the range of aerobiological sampling, improve positional accuracy of sampling paths, and enable coordinated flight with multiple UAVs sampling at different altitudes. © 2008 Wiley Periodicals, Inc.