The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridized in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG)n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.