Positron emission tomography (PET) imaging with 18F-labeled α-methyl-substituted amino acids exerts significant influence on differential diagnosis of malignant tumors and tumor-like lesions. Exclusive uptake via L-type amino acid transporter 1 (LAT1), a tumor-specific transporter, accounts for their excellent tumor specificity and low background accumulation. However, further refinement and optimization in their tumor accumulation and pharmacokinetics are sorely needed. To address these issues, we newly designed 18F-labeled α-methyl-phenylalanine (18F-FAMP) regioisomers (2-, 3-, or 4-18F-FAMP) and stereoisomers (L- or D-form), and we comprehensively evaluated their potential as tumor-imaging agents. 18F-FAMPs were prepared from α-methyl phenylalanine by electrophilic radiofluorination and purified by reversed-phase HPLC. In biodistribution studies on normal mice, L-2-18F-FAMP and the three D-18F-FAMPs showed faster blood clearance and lower renal accumulation than L-3-18F-FAMP or L-4-18F-FAMP. In LS180 human colorectal cancer cell line xenograft mice, L-2-18F-FAMP exhibited significantly higher tumor accumulation than the D-18F-FAMPs or a clinically relevant tracer, L-3-18F-α-methyl-tyrosine (18F-FAMT) (p < 0.05). The renal accumulation levels of L-2-18F-FAMP were significantly lower than that of 18F-FAMT (p < 0.01). LAT-1 specificity of L-2-18F-FAMP was validated in the cellular uptake studies. The PET imaging with L-2-18F-FAMP clearly visualized the tumor as early as 1 h after injection, and the high tumor accumulation level was retained for 3 h. These findings suggest that L-2-18F-FAMP constitutes a potential PET tracer for tumor-specific imaging.