Type III secretion system (T3SS) is an important virulence system in Gram-negative bacteria. In this investigation, different environmental conditions that regulate the expression of T3SS genes in Yersinia ruckeri were investigated aimed at obtaining a better understanding about its modulation after various environmental challenges. Four isolates of Y. ruckeri CSF007-82, ATCC29473, A7959-11 and YRNC10 were cultivated under the diverse in vitro challenges iron depletion, high salt, low pH and in the presence of fish serum or in the fish cell culture (Chinook Salmon Embryo - CHSE). The transcriptional modulation of the chromosomal genes ysaV, ysaC, ysaJ and prgH of ysa were investigated using quantitative real-time PCR. The expression of prgH, ysaV, ysaC and ysaJ was differentially expressed in all four strains under evaluation. The highest gene expression levels were observed for Y. ruckeri YRNC10 AN after addition of 0.3 M NaCl in Luria Bertani broth. The results obtained from this study provide initial insights into T3SS responses in Y. ruckeri, which pave the way for further studies aimed at expanding our knowledge on the functional roles of the T3SS genes in Y. ruckeri.