In this paper, the protocol which we have developed to get satisfactory spreads of Amoeba proteus mitotic chromosomes is presented, and the process of karyotyping this amoeba species is described. This protocol allows obtaining of extended chromosomic with repeatable chromomeres pattern in individual chromosomes. We have shown that metaphase in «B»-strain amoebae (one of the type strains of A. proteus in the Amoebae Strains Collection at the Institute of Cytology) contains 27 chromosome pairs with homologous chromomeric pattern. It is ascertained that chromomeric pattern is chromosome-specific feature. The bank of images of DAPI- and YoYo1-banded individual metaphase chromosomes of «B»-strain of A. proteus is presented.