Human normal spermatozoa present a specific chromatin organization, illustrated particularly by the non-random chromosome positioning. Spermatozoa with large vacuoles, described using motile sperm organelle morphology organization (MSOME), are associated with nuclear alterations, such as abnormal chromatin condensation and aneuploidy. To question a probable association between large nuclear vacuoles and chromatin disorganization, we evaluated chromosomes X, Y and 18 topography in normal spermatozoa (NS) compared with spermatozoa with large vacuoles (SLV). After centrifugation on a gradient density system, 229 NS (spermatozoa presenting a normal nuclear shape and a vacuole area <6.5% of head area) from 10 normal semen samples and 221 SLV (spermatozoa presenting a vacuole area >13% of head area) from 10 semen samples with teratozoospermia were selected using MSOME. A three-colour FISH was carried out using α-satellite centromeric probes for chromosomes X, Y and 18. For each chromosome, longitudinal and spatial positioning of centromeres was analysed. Distribution of each chromosome was non-random in NS and in SLV, whatever the methodology used. Using longitudinal positioning, distribution of chromosome 18 and chromosome Y centromeres did not differ significantly between SLV and NS. On the contrary, chromosome X centromeres were more frequently positioned in the posterior region of sperm nucleus in SLV (p = 0.01). Considering spatial positioning, distributions differed significantly between SN and SLV for chromosome Y (p = 0.02) and chromosome 18 (p < 10−4) and marginally for chromosome X (p = 0.08). Our study concluded to a modification in chromosomes X, Y and 18 centromere topography between NS and SLV, representing a novel and supplementary evidence to argue chromatin disorganization in SLV.