Abstract We investigated the nucleotide variation of a non-coding, chloroplast DNA (cpDNA) region to infer relationships among Tunisian fig cultivars. In this study, we examine the level of genetic diversity and its distribution using sequences of the trnL and trnF genes intergenic spacer. The non-coding region displays 28 substitution sites. Insertions and deletions involving 6 sites were found. By using the Kimura-2 method, nucleotide sequences have been aligned using the MEGA program to calculate pairwise divergence of trnL-trnF spacer sequences between cultivars. The size of this non-coding region varied from 430 to 464 bases. The relatively high A + T values (63.7–64.4%) of trnL-trnF intergenic spacer in Ficus carica may explain the high proportion of the identified transversions (ti/tv = 0.9). These results suggest the occurrence of nucleotide diversity with a large variation level of chloroplast non-coding region. The analysed data illustrate a considerable level of variability in the genetic pool of the local germplasm. In fact, relationships inferred from the cpDNA analysis suggest several clades, which do not show geographical correspondence. Fourteen haplotypes were detected among 20 individuals examined, yielding a haplotype diversity of 0.983 and a high level of nucleotide diversity (0.0100). The observed variation pattern of plastid DNA provides evidence that the fig germplasm has been undergoing rapid expansion. Neutrality tests rejected the neutrality assumption in the total sample. The cytoplasm variability indicates a narrow genetic base in the cultivated common fig. Despite the high level value of the apparent diversity, we may conclude that fig chloroplast genome provides a new conceptual and practical opportunity to evaluate genetic diversity and to identify local cultivars, making it a valuable source to include into potential breeding programs.