Two [18F]fluoroalkyl substituted amino acids differing only by the presence or absence of a methyl group on the α-carbon, (S)-2-amino-7-[18F]fluoro-2-methylheptanoic acid ((S)-[18F]FAMHep, (S)-[18F]14) and (S)-2-amino-7-[18F]fluoroheptanoic acid ((S)-[18F]FAHep, (S)-[18F]15), were developed for brain tumor imaging and compared to the well-established system L amino acid tracer, O-(2-[18F]fluoroethyl)-l-tyrosine ([18F]FET), in the delayed brain tumor (DBT) mouse model of high-grade glioma. Cell uptake, biodistribution, and PET/CT imaging studies showed differences in amino acid transport of these tracer by DBT cells. Recognition of (S)-[18F]15 but not (S)-[18F]14 by system L amino acid transporters led to approximately 8-10-fold higher uptake of the α-hydrogen substituted analogue (S)-[18F]15 in normal brain. (S)-[18F]15 had imaging properties similar to those of (S)-[18F]FET in the DBT tumor model while (S)-[18F]14 afforded higher tumor to brain ratios due to much lower uptake by normal brain. These results have important implications for the future development of α-alkyl and α,α-dialkyl substituted amino acids for brain tumor imaging.