African lions (Panthera leo) are threatened across their natural range. However, the Kgalagadi Transfrontier Park (KTP) is a stronghold for the species. A population assessment in 2010 observed a skew in the sex structure with a greater proportion of males (56%) which raised concerns about the long-term sustainability of the greater KTP lion population. A key indicator of how a population responds to changes in population structure is population size. We conducted an intensive lion census between 2013 and 2015 in the southwestern KTP (14 250 km²) driving 49 784 km over 317 sampling days, which resulted in 1162 lion sightings. Registering the population through individual identification (n = 261) provided a benchmark against which other techniques could be measured and for a non-invasive marking technique. Open-population mark-recapture provided the most precise estimate of population size (n = 246; 95%CI: 237–256). Track indices (n = 242; 95%CI: 176–307) provided a similar best estimate, but were imprecise. Minimum-known-alive calculations (n = 145) provided a gross underestimate. All the methods used in this study indicate a larger lion population size than previous estimates.