AbstractLeishmaniases affect millions of people around the world, caused by Leishmania parasites. Leishmania are transmitted by female sandflies from Phlebotominae subfamily during their blood meals. In mammals, promastigotes are phagocytosed mainly by macrophages, differentiate into amastigotes and multiply. For entry and survival in macrophages, Leishmania uses virulence factors such as surface glycoconjugates. Sialic acids (Sias) are found in terminal portions of glycoconjugates and play important roles in human pathogens. The importance of Sias was explored only in L. (L.) donovani, associated with visceral leishmaniasis in Africa, Asia and Europe. Thus, the aim of this study was to characterize Sias of Leishmania (L.) amazonensis and Leishmania (L.) infantum, related to cutaneous and visceral leishmaniasis in South America, respectively. For that, we analyzed by HPLC‐FLD the Sias of promastigotes of L. (L.) amazonensis LV79 and two L. (L.) infantum strains, and of L. (L.) amazonensis axenic amastigotes and amastigotes from paw lesions of infected mice. To evaluate Sias importance in promastigotes, we treated stationary phase parasites with sialidase and infected murine and human macrophages. We detected N‐Acetylneuraminic Acid in promastigotes of all strains, with greater abundance in L. (L.) infantum. We identified N‐Acetylneuraminic Acid and N‐Glycolylneuraminic acid in amastigotes recovered from paw lesion, but only N‐Acetylneuraminic Acid in axenic amastigotes. Promastigotes treated with sialidase infected less macrophages than parasites displaying total Sias. Our results demonstrate that Sias vary between Leishmania species and between L. (L.) amazonensis life stages and plays an important role in macrophage infection by L. (L.) amazonensis and L. (L.) infantum.