Our comprehension of avian karyotypes still needs to be improved, especially for Suliform birds. To enhance understanding of chromosomal evolution in this order, we conducted conventional and molecular cytogenetic analysis in five species, named , , (Sulidae), (Fregatidae), and (Phalacrocoracidae). The diploid chromosome number for and was established as 2 = 76 in males, and 2 = 75 in females, but . displayed a karyotype of 2 = 76 chromosomes in males. The disparity in diploid chromosome numbers between male and female is due to a multiple sex chromosome system of the ZZZZ/ZZW type. We propose that the emergence of this multiple-sex chromosome system resulted from a Robertsonian translocation involving the W chromosome and the smallest microchromosome. exhibited a diploid number 76 (2 = 76), while displayed a diploid number of 74 (2 = 74) in both sexes. The ribosomal cluster was located in one microchromosome pair in , , , and and in four pairs in . Our findings provide evidence of a conserved multiple-sex chromosome system within the genus, shedding light on the high karyotype diversity in Suliformes.