The critically endangered Wollemia nobilis W.G. Jones, K.D. Hill & J.M. Allen is endemic to Wollemi National Park north of Sydney (Australia). All known wild individuals are restricted to four sites in a single canyon system. W. nobilis can reproduce sexually but at all sites individual clumps can be multi-stemmed from a common base. In the first genetic study of this species, no genetic variation was found across multiple genetic marker types representing hundreds of nuclear loci, indicating this species is characterised by very low genetic variation. In this study we searched for variation across the chloroplast using shotgun sequencing, bioinformatic extraction of chloroplast DNA and variant detection. Six chloroplast single nucleotide polymorphisms were detected, producing three chlorotypes. Chlorotype 1 is found in every individual surveyed at Sites 1 and 3, and in individuals from Sites 2 and 4. Chlorotype 2 – the most distinct chlorotype – was found in two individuals from Site 4. Chlorotype 3 consists of a single difference from Chlorotype 1 and may represent a somaclonal mutant. These findings will guide management and translocation of this critically endangered species. This study provides a practical template that is highly informative and easily applicable to other taxa in similar circumstances.