Summary Development of high‐throughput sequencing techniques has greatly benefited our understanding about microbial ecology, yet the methods producing short reads suffer from species‐level resolution and uncertainty of identification. Here, we optimize Pacific Biosciences‐based metabarcoding protocols covering the internal transcribed spacer (ITS region) and partial small subunit of the rRNA gene for species‐level identification of all eukaryotes, with a specific focus on Fungi (including Glomeromycota) and Stramenopila (particularly Oomycota). Based on tests on composite soil samples and mock communities, we propose best suitable degenerate primers, ITS9munngs + ITS4ngsUni for eukaryotes and selected groups therein and discuss the pros and cons of long read‐based identification of eukaryotes.