Horizontal gene transfer (HGT) is the transmission of genetic material between different evolutionary lineages and is believed to be an important source of genomic innovation in fungi. In this study, we searched for prokaryotic-derived HGTs in 23 fully sequenced genomes using a comprehensive phylogenomic pipeline followed by manual curation. We found strong support for 60 HGT events comprising 190 genes putatively acquired from bacteria. HGT affected all Penicillium species to various degrees. Gene duplication events happened to 3 HGT genes after the transmission. Most HGT events include genes encoding a variety of enzymes, which are associated with sugar, amino acid, and lipid metabolism. Transcriptome data from 6 Penicillium species revealed that 33 of 35 HGT genes showed expression under the conditions tested and 16 genes were differentially expressed. Our results suggest an important role for inter-domain gene transfers in shaping the genome of Penicillium fungi. • A comprehensive phylogenomic search for acquired prokaryotic genes in the genomes of 23 Penicillium species was performed. • A set of 60 HGT events were identified to have been acquired from bacteria. • HGT genes encode a variety of enzymes which are associated with sugar, amino acid, and lipid metabolism. • HGT genes are expressed under different cultivation conditions, suggesting that they are functional. [ABSTRACT FROM AUTHOR]