Dual-color fluorescence in situ hybridization (FISH) was performed to study the simultaneous localization of major (45S) and minor (5S) family ribosomal RNA genes on chromosomes of Neolissochilus hexagonolepis. The partial 45S (18S, ITS 1, 5.8S, ITS 2 and 28S) and complete 5S (coding and NTS) rDNA units were amplified, sequenced, analyzed, and mapped on the metaphase chromosomes. The complete 18S, 5.8S and partial 28S rDNAs were 1849 bp, 157 bp and 1819 bp long, respectively. Internal transcribed spacers, namely ITS 1 (828 bp) and ITS 2 (359 bp), showed significant nucleotide variations from other fish species listed in NCBI database. The 5S rDNA contained an identical coding region of 120 bp and a highly divergent, non-transcribed 81-bp spacer. The specimens of N. hexagonolepis showed six bright fluorescent signals of 18S, while the 5S signals were present only on one pair of chromosomes. Subsequent analyses between conventional Ag-NORs and 18S rDNA FISH strongly suggested the possible inactivation of one pair of NORs that was localized at a telomeric position of a submetacentric chromosome. The sequencing and chromosomal localization of 45S and 5S rDNAs may serve as a useful genetic marker in taxonomic classification as well as in phylogenetic and evolutionary studies.