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RNA sequencing (RNA‐Seq) is popular for measuring gene expression in non‐model organisms, including wild populations. While RNA‐Seq can detect gene expression variation among wild‐caught individuals and yield important insights into biological function, sampling methods can also affect gene expression estimates. We examined the influence of multiple technical variables on estimated gene expression in a non‐model fish, the westslope cutthroat trout (Oncorhynchus clarkii lewisi), using two RNA‐Seq library types: 3′ RNA‐Seq (QuantSeq) and whole mRNA‐Seq (NEB). We evaluated effects of dip netting versus electrofishing, and of harvesting tissue immediately versus 5 min after euthanasia on estimated gene expression in blood, gill, and muscle. We found no significant differences in gene expression between sampling methods or tissue collection times with either library type. When library types …