Flexible polypeptide linkers composed of glycine and serine are important components of engineered multidomain proteins. We have previously shown that the conformational properties of Gly-Gly-Ser repeat linkers can be quantitatively understood by comparing experimentally determined F?rster resonance energy transfer (FRET) efficiencies of ECFP-linker-EYFP proteins to theoretical FRET efficiencies calculated using wormlike chain and Gaussian chain models. Here we extend this analysis to include linkers with different glycine contents. We determined the FRET efficiencies of ECFP-linker-EYFP proteins with linkers ranging in length from 25 to 73 amino acids and with glycine contents of 33.3% (GSSGSS)， 16.7% (GSSSSSS)， and 0% (SSSSSSS). The FRET efficiency decreased with an increasing linker length and was overall lower for linkers with less glycine. Modeling the linkers using the WLC model revealed that the experimentally observed FRET efficiencies were consistent with persistence lengths of 4.5， 4.8， and 6.2 ? for the GSSGSS， GSSSSS， and SSSSSS linkers， respectively. The observed increase in linker stiffness with reduced glycine content is much less pronounced than that predicted by a classical model developed by Flory and co-workers. We discuss possible reasons for this discrepancy as well as implications for using the stiffer linkers to control the effective concentrations of connected domains in engineered multidomain proteins.