Although spine variation within cacti species or populations is assumed to be large, the minimum sample size of different spine anatomical and morphological traits required for species description is less studied. There are studies where only 2 spines were used for taxonomical comparison amnog species. Therefore, the spine structure variation within areoles and individuals of one population of Gymnocalycium
kieslingii
subsp.
castaneum (Ferrari) Slaba was analyzed. Fifteen plants were selected and from each plant one areole from the basal, middle and upper part of the plant body was sampled. A scanning electron microscopy was used for spine surface description and a light microscopy for measurements of spine width, thickness, cross-section area, fiber diameter and fiber cell wall thickness. The spine surface was more visible and damaged less in the upper part of the plant body than in the basal part. Large spine and fiber differences were found between upper and lower parts of the plant body, but also within single areoles. In general, the examined traits in the upper part had by 8–17% higher values than in the lower parts. The variation of spine and fiber traits within areoles was lower than the differences between individuals. The minimum sample size was largely influenced by the studied spine and fiber traits, ranging from 1 to 70 spines. The results provide pioneer information useful in spine sample collection in the field for taxonomical, biomechanical and structural studies. Nevertheless, similar studies should be carried out for other cacti species to make generalizations. The large spine and fiber variation within areoles observed in our study indicates a very complex spine morphogenesis.