The 5' untranslated regions (UTR) of chloroplast mRNAs often contain regulatory sequences that control RNA stability and/or translation. The petD chloroplast mRNA in Chlamydomonas reinhardtii has three such essential regulatory elements in its 362-nt long 5' UTR. To further analyze these elements, we compared 5' UTR sequences from four Chlamydomonas species (C. reinhardtii, C. incerta, C. moewusii and C. eugametos) and five independent strains of C. reinhardtii. Overall, these petD 5' UTRs have relatively low sequence conservation across these species. In contrast, sequences of the three regulatory elements and their relative positions appear partially conserved. Functionality of the 5' UTRs was tested in C. reinhardtii chloroplasts using beta-glucuronidase reporter genes, and the nearly identical C. incerta petD functioned for mRNA stability and translation in C. reinhardtii chloroplasts while the more divergent C. eugametos petD did not. This identified what may be key features in these elements. We conclude that these petD regulatory elements, and possibly the corresponding trans-acting factors, function via mechanisms highly specific and surprisingly sensitive to minor sequence changes. This provides a new and broader perspective of these important regulatory sequences that affect photosynthesis in these algae.