[NiFe] hydrogenases reversibly catalyze hydrogen oxidation and proton reduction at a Ni-Fe active site coordinated by four cysteine (Cys) residues in two CXXC motifs within the catalytic large subunit. A subset of these enzymes contains selenocysteine (Sec) in place of Cys in the C-terminal CXXC motif, forming [NiFeSe] hydrogenases with enhanced oxygen tolerance and hydrogen production activity. However, such Sec-containing variants have been reported only in Groups 1 and 3 [NiFe] hydrogenases. Here, we present a comprehensive dataset of Group 4 [NiFeSe] hydrogenases based on a genomic survey combined with UGA stop-codon read-through analysis. The dataset comprises 47 non-redundant protein sequences from nine bacterial phyla. Sec substitutions were exclusively identified in the N-terminal CXXC motif, including 27 CXXU-, 4 UXXC-, and 16 UXXU-type sequences, indicating the emergence of non-canonical Sec-containing motifs in Group 4. Phylogenetic analysis revealed a sporadic distribution across three distinct lineages, indicating at least three independent origins of Sec-containing variants from Cys-type ancestors. Such substitutions were found across diverse ecosystems. Genomic context analysis further suggests that these Sec-containing enzymes form energy-converting complexes, similar to those formed by other Group 4 enzymes. The Sec-containing variants co-occur with Sec biosynthesis genes and a conserved guanine residue in the apical loop of Sec insertion sequence elements. These findings provide new insights into the evolution of Sec utilization in [NiFe] hydrogenases.