We used an existing conservation opportunity area (OA) data layer for four contiguous ecological subsections within the Ozark Highlands to quantitatively evaluate the influence of conservation targets and assessment region size on conservation priorities. OAs are natural and seminatural land-cover patches that are away from roads and away from patch edges. To evaluate the influence of targets, we assigned a priority score to each OA polygon for each of five different conservation targets, including land-cover patch size, landform representation, target vertebrate richness, target breeding bird richness, and target land cover. The top-scoring OAs for each target were added to an OA selection set for that target until 50% of the study area was chosen. These five OA selection sets were overlain to quantify overlap in priorities. Only 1.6% of the study area, or 2.1% of all OA polygons, was selected by all five targets. To evaluate the influence of assessment region size, we compared results of priority ranking of OAs relative to the entire study area against a merged set of priority rankings established separately relative to each of the four subsections within the study area. When high-priority OAs were added until 25% of the region was within the selection set for each of the five targets, the sets based on the whole study area versus each subsection evaluated separately overlapped from 45.4% to 81.9%. Thus, perceived priorities of conservation assessments are strongly influenced both by the targets that are evaluated and by the size of the assessment region.