Chloroplast-localized small heat-shock proteins (Cp-sHSP) protect Photosystem II and thylakoid membranes during heat and other stresses, and Cp-sHSP production levels are related to plant thermotolerance. However, to date, a paucity of Cp-sHSP sequences from C4 or CAM species, or from other extremely heat-tolerant species, has precluded an examination to determine if Cp-sHSP genes or proteins might differ among plants with photosynthetic pathways or between heat-sensitive and heat-tolerant species. To investigate this, we isolated and characterized novel Cp-sHSP genes in four plant species: two moderately heat-tolerant C4 species, Spartina alterniflora (monocot) and Amaranthus retroflexus (eudicot), and two very heat-tolerant CAM species, Agave americana (monocot) and Ferocactus wislizenii (eudicot) (respective genes: SasHSP27.12, ArsHSP26.43, AasHSP26.85 and FwsHSP27.52) by PCR-based genome walking and cDNA RACE. Analysis of these Cp-sHSPs has confirmed the presence of conserved domains common to previously examined species. As expected, the transit peptide was found to be the most variable part of these proteins. Promoter analysis of these genes revealed differences in CAM versus C3 and C4 species that were independent of a general difference between monocots and eudicots observed for the entire protein. Heat-induced gene and protein expression indicated that Cp-sHSP protein levels were correlated with thermotolerance of photosynthetic electron transport, and that in most cases protein and transcript levels were correlated. Thus, available evidence indicates little variation in the amino acid sequence of Cp-sHSP mature proteins between heat-sensitive and -tolerant species, but that variation in Cp-sHSP protein production is related to heat tolerance or photosynthetic pathway (CAM vs. C3 and C4) and is driven by promoter differences. Key message We isolated and characterized four novel Cp-sHSP genes with promoters from wild plants, analysis has shown qualitative and quantitative interspecific variations in Cp-sHSPs of C3, C4, and CAM plant thermotolerance.