The analysis of transcripts in situ provides valuable spatial information on gene expression at tissue-level and/or subcellular resolution. Approaches involving hybridization or sequencing have been developed, but these have been limited to characterizing the expression of only a few genes simultaneously. Lee et al. now present a new method of fluorescent in situ RNA sequencing (FISSEQ) that is applicable to various sample types from cultured cells to tissue sections and whole-mount embryos. All steps — reverse transcription, amplification and fluorescence-based sequencing — are carried out across the transcriptome at the cellular location of each transcript. Such an approach provides opportunities for spatially annotated global expression analyses.