The Matjes River Rock Shelter, in the Plettenberg Bay region of South Africa, has a large collection of archaeological cranial remains representing 8,000 years of occupation. Forty-three adult crania from the Matjes River collection are studied here, many which have not previously been published. These crania are compared through multivariate analysis with other dated crania from Fish Hoek, Cape Flats, Elands Bay, Port Elizabeth and Kenkelbosch, and with archaeological crania from five ecological biomes, Forest, Fynbos, Nama Karoo, Savanna, and Succulent Karoo biomes in southern Africa. Historical southern African populations, KhoeSan (31) and South African Negroid (120) crania, are also used to compare the affinities of the Ma~es River individuals. The sample sizes within this study are small and an attempt has been made to address this issue by the use of multiple analytical methods and multiple 'tests' of variable sets to improve the statistical significance of results. This study, incorporating the largest collection of Matjes River individuals, uses principal components analysis to show the individuals to be a single biological population and illustrates with Euclidean distance statistic strong morphological similarities between the individuals. From principal component analysis, and discriminant function analysis there is no determination of morphological differences between the crania on the basis of geographical or environmental differentiation. Overall the KhoeSan crania in this study show homogeneity in form. Holocene gracilisation is seen in the dated crania through principal components analysis. The temporal changes found also indicate a change over time relating to nasal height measurements, while Spearman's rank correlation coefficient found no temporal shape changes occurring between the dated samples. Interestingly, discriminant analysis classification found non-KhoeSanid morphology is represented by several dated individuals as early as 9,000 years BP. This indication of non-KhoeSanid morphology may hint at possible admixture of the KhoeSan population with outsiders, but is a stronger indicator that origins of KhoeSan craniofacial patterns are not yet fully understood.