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The two major diphospho inositol phosphates from the axenic strain Dictyostelium discoideumAX2 were previously investigated and identified as 6‐PP‐InsP5and 5,6‐bis‐PP‐InsP4. In order to examine whether these findings are representative of Dictyostelids in general, five non‐axenic wild‐type species of Dictyosteliumand two of Polysphondyliumwere studied. It was found that all of the Dictyosteliumspecies exhibit similar patterns of diphospho inositol phosphates. By contrast, both of the Polysphondyliumspecies contain 5‐PP‐InsP5as the predominant isomer. Besides 5,6‐bis‐PP‐InsP4, a new bis‐PP‐InsP4was detected in Polysphondylium. This compound is either 1,5‐bis‐PP‐InsP4or its corresponding enantiomer 3,5‐bis‐PP‐InsP5. The structures were elucidated by two‐dimensional 1H‐1H and 1H‐31P NMR analysis. Additionally, they were confirmed using a specific 6‐PP‐InsP5‐5‐kinase from D. discoideumAX2 as an enantio‐specific tool and enantiomerically pure reference standards.