Iodine (I) is considered a beneficial element or even micronutrient for plants. The aim of this study was to determine the molecular and physiological processes of uptake, transport, and metabolism of I applied to lettuce plants. KIO3, KIO3+ salicylic acid, 5-iodosalicylic acid and 3,5-diiodosalicylic acid were applied. RNA-sequencing was executed using 18 cDNA libraries constructed separately for leaves and roots from KIO3, SA and control plants. De novo transcriptome assembly generated 1937.76 million sequence reads resulting in 27,163 transcripts with N50 of 1638 bp. 329 differentially expressed genes (DEGs) in roots were detected after application of KIO3, out of which 252 genes were up-regulated, and 77 were down-regulated. In leaves, 9 genes revealed differential expression pattern. DEGs analysis indicated its involvement in such metabolic pathways and processes as: chloride transmembrane transport, phenylpropanoid metabolism, positive regulation of defense response and leaf abscission, and also ubiquinone and other terpenoid-quinone biosynthesis, protein processing in endoplasmic reticulum, circadian rhythm including flowering induction as well as a putative PDTHA (i.e. Plant Derived Thyroid Hormone Analogs) metabolic pathway. qRT-PCR of selected genes suggested their participation in the transport and metabolism of iodine compounds, biosynthesis of primary and secondary metabolites, PDTHA pathway and flowering induction.