Translation initiation is a key step determining protein synthesis. Studies have uncovered a number of alternative translation initiation sites (TISs) on mammalian genes and demonstrated their roles in reshaping the proteome. However, the extent to which alternative TISs affects gene expression across plants remains largely unclear. Here, by profiling initiating ribosome positions, we globally identified in vivo TISs in tomato and Arabidopsis and found thousands of genes with greater than one TIS. Of the identified TISs, >19% and >21% were located at unannotated ATG and non-ATG sites, in which CTG and ACG were most frequently observed, a phenomenon also found in mammals. In addition, while the alternative TISs were significantly found in both orthologous genes, the TIS site sequences were not conserved, suggesting the conservation of alternative initiation mechanisms but flexibility in employing TIS sites. Intriguingly, unlike the role of upstream ATG TISs in translational repression, the presence of upstream non-ATG TISs was significantly correlated with higher transcript abundances, a pattern observed across plants but not in mammals. Also, the generation of proteins with diverse N-terminals through the use of alternative TISs contributes to differential subcellular localization, as mutating alternative TISs resulted in the loss of organelle localization. Our findings uncovered the hidden coding potential of plant genomes and, importantly, the constraint and flexibility of translational initiation mechanisms in the regulation gene expression across plant species.