High-throughput sequencing for analysis of environmental microbial diversity has evolved vastly over the last decade. Currently the go-to method for planktonic microbial eukaryotes is short-read metabarcoding of 18S rRNA with <500 bp amplicons. However, there is a growing interest to sequence longer amplicons of ~4.5 kbp covering the rRNA operon for improving taxonomic resolution. For both methods, the choice of primers is crucial. It determines if central community members are covered, if they can be identified at a satisfactory taxonomic level, and if the obtained community profile is undistorted. We designed new primers targeting 18S and 28S rRNA based on 177,934 and 21,072 database sequences, respectively. We evaluated the primers along with eight published pairs in silico on sequence databases and metagenomics datasets. We selected ten and three pairs for short- and long-read library preparation, respectively, to compare the obtained community profile of six environmental samples with primer-unbiased metagenomics sequencing. We found that the primers provide different community impressions based on the targeted region. Of the short-read pairs, a new V6-V8 pair and the V4_Balzano pair used with a simplified PCR protocol provided good results in silico and in vitro. Less differences were observed between the long-read pairs. The long-read amplicons and also ITS1 alone provided higher taxonomic resolution than V4, however for use in high-throughput metabarcoding an extensive reference database of the regions is lacking. Together, our results represent a reference and guide for selection of robust primers for research and environmental monitoring of microbial eukaryotes.