Conservation plans may conflict when both predator and prey in the same ecological system are threatened species. In this study we present a problematic case of conflicting conservation policies involving the Arabian wolf Canis lupus arabs and two species of gazelles (Gazella gazella acaciae and Gazella dorcas), all of which are threatened in Israel. By studying genetic subdivision using faecal DNA we evaluated the policy of treating the Arabian wolves in the Negev Desert as two separate populations. We analysed 95 wolf faecal samples from 12 feeding sites c. 20 km apart. Network analysis and Bayesian clustering were used for separating populations. Mark–recapture design, rarefaction and an urn model were applied to estimate wolf population size. We found that wolves in the central and southern Negev cannot be genetically separated, and their density is similar in both regions. Our results provide a better baseline for a unified management of wolves in the Negev. We call for the consideration of other factors influencing gazelle population size before adopting drastic measures such as wolf removal.