The larvae of fish are exposed to microbes from the day of hatching until the maturation of lymphoid organs from which the fish is capable of responding the pathogens through adaptive immune system. During the maturation of lymphoid organs the phenomenon of V(D)J recombination is mediated by recombination activating genes (RAGs), which forms the marker of physiological maturity of the immune system. In the present study, the RAG-1 nucleotide sequence of 272bp was cloned partially in order to evaluate its pattern of mRNA expression during ontogenetic development and tissues of Pterophyllum scalare. However, before analysing the quantitative PCR (qRT-PCR) data of RAG-1 it was necessary to validate a suitable reference gene. For the selection of suitable internal control the commonly used housekeeping genes viz. alpha-actin (α-actin), beta-actin (β-actin), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor-alpha 1 (EF1α) were first characterized and evaluated for validation. Among the four reference genes, β-actin was found to be the most stable gene in different tissues and during various developmental stages of P. scalare. The mRNA expression of RAG-1 was detected right from the hatching day and showed an up-regulation upto 30 dph after which it started declining till the end of experiment. Moreover, the distribution of RAG-1 in different tissues exhibited its high expression level in kidney compared to other tissues. The study concludes that the complete development of lymphoid organs in P. scalare occurs at 27 to 30 dph where the mRNA expression of RAG-1 was significantly high.