Proteins localized to mitochondria by a carboxyl-terminal tail anchor (TA) play important roles in apoptosis, mitochondrial dynamics, and mitochondrial protein import. In order to reveal structural characteristics of TAs that may be important for mitochondrial targeting, we focused our attention upon the TA of the Saccharomyces cerevisiae Fis1 protein. We generated a library of Fis1p TA variants fused to the transcription factor Gal4p. By selecting for mutations within the TA permitting Gal4p to translocate to the nucleus and activate transcription, we were able to enrich for TA variants within our mutant pool which led to decreased membrane insertion. Next-generation sequencing allowed quantification of each TA variant in our mutant library both before and after selection, and high-throughput results were confirmed by microscopy-based and functional analysis of individual, reconstructed Fis1 TA mutants. Consistent with the prediction that the Fis1 TA is alpha-helical, prolines within the TA generally prevented membrane insertion. Charged residues within the hydrophobic core of the Fis1 TA were also able to perturb membrane insertion. Surprisingly, positively charged residues were much more acceptable at several positions within the Fis1 TA than negatively charged residues, providing strong, in vivo evidence that lysine and arginine can “snorkel,” or partition the nonpolar portion of their side chains into the hydrophobic region of the lipid bilayer while placing the terminal charge near the polar interface of the membrane. In further experiments, we found that lengthening or shortening the Fis1 TA by up to three amino acids did not alter its behavior or localization, arguing against a model in which TA length directs insertion into specific organelles. Finally, our data affirm previous results indicating that the charged carboxyl-terminus of Fis1p is important for localization and insertion at the mitochondrial outer membrane. Our work provides the first high-resolution analysis of an organelle targeting sequence to be generated by deep mutational scanning.