Carotenoid content is the primary determinant of fruit color that affects nutritional value and appearance in tomato. Phytoene synthase (PSY) is the key regulatory enzyme in the carotenoid biosynthesis pathway. Absent function of PSY1 in tomato fruit results in yellow flesh phenotype. We, here, report that two different transcripts, a wild-type (Psy1) and a chimeric mRNA (Psy1/Unknown), exist in a yellow-fruited tomato accession PI 114490. Psy1/Unknown is generated by joining exons from two different genes, Psy1 and an unknown gene, transcribed using both complementary DNA strands. The Psy1 shows low expression in the fruit of PI 114490, while the expression of Psy1/Unknown in the fruit of PI 114490 shows the same pattern as Psy1 in red fruit. The PSY1/Unknown has a lower function than PSY1 in a bacterial expression system. Coincidence of one single-nucleotide polymorphism (SNP) in the fourth intron and one simple sequence repeat (SSR) with 19 AT repeats in the downstream sequence of Psy1 gene with Psy1/Unknown in a set of yellow-fruited tomato lines indicates that Psy1/Unknown might be caused by the SNP and/or SSR. One possible explanation of these observations is trans-splicing. Severely reduced Psy1 transcript caused by Psy1/Unknown results in low accumulation of carotenoid and yellow flesh in PI 114490.