BackgroundThe regulation of gene expression during tissue development is very complex. A key mechanism of gene regulation is the recognition of regulatory motifs, also known as cis-regulatory elements (CREs), by various proteins in gene promoter regions. Localization of these motifs near the transcription start site (TSS) or translation start site (ATG) is crucial for transcription initiation and rate. Transcription levels of individual genes, regulated by these motifs, can vary significantly across tissues and developmental stages, especially in processes like sexual reproduction. However, the precise localization and visualization of regulatory motifs in relation to gene expression in specific tissues can be challenging.ResultsHere, we introduce a program called GOLEM (Gene regulatOry eLEMents) which enables users to precisely locate any motif of interest with respect to TSS or ATG within the relevant plant genomes across the plant Tree of Life (Marchantia, Physcomitrium, Amborella, Oryza, Zea, SolanumandArabidopsis). The visualization of the motifs is performed with respect to the transcript levels of particular genes in leaves and male reproductive tissues and can be compared with genome-wide distribution regardless of the transcription level. Additionally, genes with specific CREs at defined positions and high expression in selected tissues can be exported for further analysis. GOLEM’s functionality is illustrated by its application to conserved motifs (e.g. TATA-box, ABRE, I-box, and TC-element), as well as to male gametophyte-related motifs (e.g. LAT52, MEF2, ARR10_core, and DOF_core).ConclusionGOLEM is a freely available tool (https://golem.ncbr.muni.cz) for tracking the precise localization and distribution of any CREs of interest in plant gene promoters.Graphical abstract