The order Gobiiformes is made up of more than 2200 species, representing one of the most diverse groups among teleost fishes. The biological causes for the tachytelic karyotype evolution of the gobies have not yet been fully studied. Here we expanded cytogenetic data for the Eleotridae family, analyzing the neotropical species Dormitator maculatus, Eleotris pisonis, Erotelis smaragdus, and Guavina guavina. In addition, a meta-analytical approach was followed for elucidating the karyotype diversification versus biological aspects (habitat and egg type) of the Gobiiformes. The species E. smaragdus and E. pisonis present 2n = 46 acrocentric chromosomes (NF = 46), D. maculatus 2n = 46 (36sm + 4st + 6a; NF = 86), and G. guavina, the most divergent karyotype, with 2n = 52 acrocentric chromosomes (NF = 52). Besides numeric and structural diversification in the karyotypes, the mapping of rDNAs and microsatellites also showed noticeable numerical and positional variation, supporting the high chromosomal evolutionary dynamism of these species. In Gobiiformes, karyotype patterns which are more divergent from the basal karyotype (2n = 46a) are associated with characteristics less effective to dispersion, such as the benthic habit. These adaptive characteristics, connected with the organization of the repetitive DNA content in the chromosomes, likely play a synergistic role in the remarkable karyotype diversification of this group.