Next generation sequencing (NGS) technology transformed Leishmania genome studies and became an indispensable tool for Leishmania researchers. Recent Leishmania genomics analyses facilitated the discovery of various genetic diversities including single nucleotide polymorphisms (SNPs), copy number variations (CNVs), somy variations, and structural variations in detail and provided valuable insights into the complexity of the genome and gene regulation. Many aspects of Leishmania NGS analyses are similar to those of related pathogens like trypanosomes. However, the analyses of Leishmania genomes face a unique challenge because of the presence of frequent aneuploidy. This makes characterization and interpretation of read depth and somy a key part of Leishmania NGS analyses because read depth affects the accuracy of detection of all genetic variations. However, there are no general guidelines on how to explore and interpret the impact of aneuploidy, and this has made it difficult for biologists and bioinformaticians, especially for beginners, to perform their own analyses and interpret results across different analyses. In this guide we discuss a wide range of topics essential for Leishmania NGS analyses, ranging from how to set up a computational environment for genome analyses, to how to characterize genetic variations among Leishmania samples, and we will particularly focus on chromosomal copy number variation and its impact on genome analyses.