In continuation of our previous research we carried out the karyological investigation of 53 populations of four Aegilops species (A. geniculata, A. triuncialis, A. ventricosa, and A. neglecta) sampled in different eco-geographical habitats in Algeria. The genetic variability of the chromosomal DNA loci of the same collection of Aegilops is highlighted by the Fluorescence In Situ Hybridization technique (FISH) using three probes: 5S rDNA, 45S rDNA, and repetitive DNA (pSc119.2). We found that the two rDNA loci (5S and 45S) hybridized with some chromosomes and showed a large genetic polymorphism within and between the four Aegilops species, while the repetitive DNA sequences (pSc119.2) hybridized with all chromosomes and differentiated the populations of the mountains with a humid bioclimate from the populations of the steppe regions with an arid bioclimate. However, the transposition of the physical maps of the studied loci (5S rDNA, 45S rDNA, and pSc119.2) with those of other collections revealed the existence of new loci in Aegilops from Algeria.