The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ(5)- and Δ(6)-elongases of Marchantia polymorpha as well as the Δ(6)-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ(9)-, Δ(6)-C18, and Δ(5)-C20 LC-PUFAs, whereas only Δ(9)- and Δ(6)-C18 LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C20 fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C20 substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.