For biodiversity protection to play a persuasive role in land‐use planning, conservationists must be able to offer objective systems for ranking which natural areas to protect or convert. Representing biodiversity in spatially explicit indices is challenging because it entails numerous judgments regarding what variables to measure, how to measure them, and how to combine them. Surprisingly few studies have explored this variation. Here, we explore how this variation affects which areas are selected for agricultural conversion by a land‐use prioritization model designed to reduce the biodiversity losses associated with agricultural expansion in Zambia. We first explore the similarity between model recommendations generated by three recently published composite indices and a commonly used rarity‐weighted species richness metric. We then explore four underlying sources of ecological and methodological variation within these and other approaches, including different terrestrial vertebrate taxonomic groups, different species‐richness metrics, different mathematical methods for combining layers, and different spatial resolutions of inputs. The results generated using different biodiversity approaches show very low spatial agreement regarding which areas to convert to agriculture. There is little overlap in areas identified for conversion using previously published indices (mean Jaccard similarity, Jw, between 0.3 and 3.7%), different taxonomic groups (5.0% < mean Jw < 13.5%), or different measures of species richness (15.6% < mean Jw < 33.7%). Even with shared conservation goals, different methods for combining layers and different input spatial resolutions still produce meaningful, though smaller, differences among areas selected for conversion (40.9% < mean Jw < 67.5%). The choice of taxonomic group had the largest effect on conservation priorities, followed by the choice of species richness metric, the choice of combination method, and finally the choice of spatial resolution. These disagreements highlight the challenge of objectively representing biodiversity in land‐use planning tools, and present a credibility challenge for conservation scientists seeking to inform policy making. Our results suggest an urgent need for a more consistent and transparent framework for designing the biodiversity indices used in land‐use planning, which we propose here.