Argan trees are normally endemic to Morocco and Algeria, but hundreds of argan trees exist in Tunisia, some introduced from Morocco and some from unknown origins. The aim of the present study was to evaluate the genetic, morphological, and biochemical diversity of the argan trees in Tunisia. In this study, we used morphometric data collected from vegetative tissue, as well as pomological characteristics related to fruits, stones, and kernels. Genetic variation in 60 trees of Tunisian Argania spinosa L. was estimated using inter-simple sequence repeats (ISSRs). Mutation screening and genotyping by high-resolution melting (HRM) was performed to detect delta-6-desaturase (D6D) variants in the tested individuals, and finally fatty acid analysis of argan leaves with gas chromatography (GC) was performed. The plant materials used in this study originated from four different sites in Tunisia. Analysis of morphological characteristics showed large variability both within and between the studied collections. The analysis of ISSR polymorphisms gave information about the diversity within and between populations. HRM analysis showed that all 60 argan individuals were grouped into 10 different categories. The results of the gas chromatography analysis showed that the presence of omega-3 fatty acids EPA and DHA was noticeable in some argan leaves.