The tomato leafminer, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is one of the most important pests of tomato. With the purpose of developing environmentally friendly control tactics such as the inherited sterility (IS) technique against this species, it is essential to understand its genetics and biology. In this paper we analyzed the karyotype, sperm morphology and sperm ratio in wild-type and X-ray irradiated individuals of T. absoluta. The diploid chromosome number of T. absoluta was 2n = 58 including the pair of sex chromosomes: ZZ in males and WZ in females, which were the largest elements of the complement. Irradiation of pupae in an X-ray machine with a dose of 200 Gy generated various types of chromosomal rearrangements including translocations and fragmentations, resulting in altered chromosome numbers. The analysis of spermatozoa in T. absoluta revealed a significant morphological difference between apyrene and eupyrene sperm bundles. Irradiation with X-ray doses of 100, 150, 200 and 250 Gy did not have a significant effect on the apyrene to eupyrene sperm ratio. However, males irradiated with 300 Gy produced significantly more apyrene sperm than non-irradiated males. All the doses applied influenced the morphology of eupyrene sperm bundles. The modified eupyrene sperm bundles could be used as a bioindicator during the monitoring of an IS program after the release of irradiated males. We found that the modified eupyrene spermatozoa were transferred to the bursae copulatrices of the females. Males treated with 200 Gy transferred a greater proportion of modified eupyrene sperm than untreated males. The results presented herein provide essential information on the cytology of T. absoluta, which is required to evaluate the quality of the released insects, and for better understanding and application of IS against this economically important pest.