The ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions are commonly used to identify fungi and other eukaryotic taxa in amplicon sequencing. The highly conserved rRNA regions flanking the ITS need to be trimmed before being used for taxonomic assignment. The Python software package ITSxpress rapidly trims single-end or paired-end sequences in FASTQ format for use in amplicon sequence variant clustering methods like DADA2. This new major release of ITSxpress improves the paired-end merging method, simplifies installation of the QIIME 2 ITSxpress plugin, removes major dependencies, adds use cases, and is compatible with newer compression formats. This paper discusses the modifications to ITSxpress that improve the output and user experience, leading to a major version increase.