Crematogaster Lund, 1831 is a speciose ant genus globally distributed and easily recognizable. Although biogeographical theories explain some variation among Neotropical Crematogaster, several taxonomical issues remain unresolved. While cytogenetic approaches can help to delimit species, cytogenetic data are only available for 18 taxa. In this study, classical and molecular cytogenetic analyses were performed on five Crematogaster species from the Brazilian Amazon to identify species-specific patterns. Two different cytotypes, both with 2 n = 22 chromosomes were observed in Crematogaster erecta Mayr, 1866, suggesting the presence of cryptic species, although with different karyotypic formulas. Crematogaster aff. erecta had 2 n = 28, while Crematogaster limata Smith, 1858, Crematogaster tenuicula Forel, 1904, and Crematogaster sp. had 2 n = 38. The telomeric motif (TTAGG) n was found in all five species, and the (TCAGG) n motif was detected in the telomeres of C. limata. This peculiar motif was also detected in the centromeric regions of C. erecta cytotype I. The microsatellite (GA) n was dispersed in the chromosomes of all species studied, which also had a single intrachromosomal rDNA site. The cytogenetic results revealed notable interspecific and intraspecific variation, which suggests different chromosomal rearrangements involved in the origin of these variations, also highlighting the taxonomic value of cytogenetic data on Crematogaster.