Background and objectivesThis study reports for the first time the variation of prolamins profiles among 51 pearl millet landraces representing seven populations prospected from contrasted bioclimatic areas of Tunisia.FindingsA total of 27 prolamins bands were detected, ranging from 11 to 245 kDa. Among them, 14 were polymorphic resulting in a polymorphism rate of 51.85%. The bands with lower molecular weights are the most frequent. The biochemical marker M25.4 exhibited the highest value of Nei genetic diversity h (0.500), Shannon information index I (0.693) and effective number of alleles per locus Ne (2). At the population level, the polymorphism rates ranged between 50% (Mahdia) and 92.86% (Hammem Jebli, Kairouan, Medenine). The population of Kairouan displayed the highest genetic diversity indices (I = 0.511 ± 0.054, h = 0.345±0.041), while the lowest levels were recorded for the population of Mahdia (I = 0.268 ± 0.076, h = 0.177 ± 0.052). AMOVA analysis indicated that the highest amount of genetic diversity was encompassed within the studied populations (89%). This was supported by low genetic differentiation (PhiPT = 0.110, P value= 0.029) and Nei genetic distance values. The classification pattern using ward dendrogram and factorial analyses revealed the distinction of two clusters with a low genetic structure based on the geographic origins of landraces.Significance and NoveltyThe detected biochemical protein polymorphism could be a highly effective tool for the genetic resources characterization of pearl millet landraces which will be further utilized in breeding investigations and taxonomic surveys of this cereal crop.This article is protected by copyright. All rights reserved.