A putative novel methanotrophic genus, Candidatus Methylumidiphilus (Methylococcales), was recently shown to be ubiquitous and one of the most abundant methanotrophic genera in water columns of oxygen-stratified lakes and ponds in boreal and subarctic areas. However, it has probably escaped detection in many previous studies that used 16S rRNA gene amplicon sequencing due to insufficient database coverage, as previously analysed metagenome-assembled genomes (MAGs) affiliated with Ca. Methylumidiphilus do not contain 16S rRNA genes. Therefore, we screened MAGs affiliated with the genus for their 16S rRNA gene sequences in a recently published lake and pond MAG data set. Among 66 MAGs classified as Ca. Methylumidiphilus (with completeness over 40% and contamination less than 5%) originating from lakes in Finland, Sweden and Switzerland as well as from ponds in Canada, we found 5 MAGs, each containing one 1532 bp sequence spanning the V1-V9 regions of the 16S rRNA gene. After removal of sequence redundancy, this resulted in 2 unique 16S rRNA gene sequences. These sequences represented 2 different putative species: Ca. Methylumidiphilus alinenensis (GenBank accession OK236221) and another unnamed species of Ca. Methylumidiphilus (GenBank accession OK236220). We suggest that including these 2 sequences in reference databases will enhance 16S rRNA gene-based detection of members of this genus from environmental samples.