We conducted in vitro transcription activities of the three zebrafish U6 putative promoters across species in cell extracts prepared from Nile tilapia (Oreochromis niloticus) and shrimps. The transcription efficiency of these putative U6 promoters in Nile tilapia cell extracts was similar to that of zebrafish cell extracts. In addition, all three zebrafish U6 snRNA promoters were able to express the shRNA in cell extracts prepared from two shrimp species, Penaeus monodon and Litopenaeus vannamei. However, the shRNA transcription products in shrimp cell extracts showed weaker signals. These U6 promoters could promote shRNA expression, suggesting that they have the potential for use for vector-based RNAi in Nile tilapia and shrimps. A putative U6 promoter would provide a powerful tool for long-term GKD in these aquaculture-related species.