The genotyping of insertion–deletion polymorphisms (Indels) has gained importance in population studies and human identification over the last few years and so has the study of the X-chromosome markers. In the present work, 245 samples from unrelated individuals from Somalia (148 males) and Iraq (58 males and 39 females) were analysed for 33 X-chromosome Indel markers. The aim was to characterize the diversity pattern in both populations, contributing at the same time to a better understanding of the genetic landscape of the Horn of Africa and the Middle East.Although intrapopulation diversity values for the Somali and Iraqi populations were similar, significant FST values were obtained between the groups. No departure from Hardy–Weinberg equilibrium was detected in the subset of 39 Iraqi women. Linkage disequilibrium analyses revealed significant association in some pairs of markers in Somalis (MID3690–MID2089; MID3719–MID2089; MID357–MID356) and Iraqis (MID3719–MID2089; MID357–MID356). Overall, statistical parameters for forensic efficiency presented high values. The results of the analyses of forensically relevant statistical parameters suggest that the 33plex system can be used for human identification and kinship testing in Somalis and Iraqis and most likely in other populations as well.