Microsporidia are obligate intracellular parasites infecting a wide range of invertebrate and vertebrate hosts including human. The spore contains a specialized organelle called the polar tube involved in host cell invasion. Three polar tube proteins (PTP1‐3) have been identified in Encephalitozoon cuniculi. The genes coding for ptp1 and ptp2 are localized on the same chromosome and are at a distance of 860 bp. A similar synteny organization is found in two other Encephalitozoon species. Comparison of the E. cuniculi genome with that of Nosema locustae, an microsporidia parasite of grasshoppers, indicates that several genes are positioned in the same order and orientation. This allowed the identification of two ORFs distant of 1117 bp, that despite divergent sequence features, encode proteins having common characteristics with Encephalitozoon PTP1 and PTP2 (signal peptide, proline‐rich internal repeats for PTP1, lysine‐rich protein for PTP2 …). To test whether they correspond to PTP, polyclonal sera were raised against two recombinant proteins expressed in E. coli. In indirect immunofluorescence, the extruded polar tubes of N. locustae spores are specifically labelled. NlPTP1 and NlPTP2 have 355 and 287 amino acids, respectively, and are only soluble in the presence of high concentrations of reducing agent.Aptp1 and ptp2 containing cluster have been also identified in Nosema grylli. PTP1 from N. locustae and N. grylli present a strong variability in the proline‐rich internal repeats, whilst PTP2s are highly conserved. It would be interesting to show whether this gene synteny conservation is related to a specific interaction between PTP1 and PTP2.