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Peroxisomes are universal eukaryotic organelles essential to plants and animals. Most peroxisomal matrix proteins carry peroxisome targeting signal type 1 (PTS1), a C‐terminal tripeptide. Studies from various kingdoms have revealed influences from sequence upstream of the tripeptide on peroxisome targeting, supporting the view that positive charges in the upstream region are the major enhancing elements. However, a systematic approach to better define the upstream elements influencing PTS1 targeting capability is needed. Here, we used protein sequences from 177 plant genomes to perform large‐scale and in‐depth analysis of the PTS1 domain, which includes the PTS1 tripeptide and upstream sequence elements. We identified and verified 12 low‐frequency PTS1 tripeptides and revealed upstream enhancing and inhibiting sequence patterns for peroxisome targeting, which were subsequently validated …