The aim of this study was to assess the effectiveness of sperm selection by single layer centrifugation (SLC) on canine sperm quality when SLC was performed before or after the cooling process, or when double SLC (before and after cooling) was performed. Twenty ejaculates from four dogs were divided into four aliquots as follows: unselected: no SLC was performed; SLC prior to cooling (SLC-PC): sperm selection was carried out before cooling; SLC after cooling (SLC-AC): sperm selection was performed after cooling; and double SLC: sperm selection was carried out before and after cooling. Sperm motility (by computer-assisted semen analysis), morphology (Diff-Quick staining), sperm membrane integrity (Vital-Test kit) and acrosome integrity (double fluorescent stain) were assessed in re-warmed semen samples. Four sperm subpopulations (sP) were detected using a pattern analysis technique (sP1: highly active, non-progressive; sP2: low velocity, highly progressive; sP3: less vigorous, poorly progressive; sP4: highly progressive motility). A higher proportion of sperm were classified as sP4 in SLC-AC samples. Most of the sperm parameters assessed showed higher values in the SLC-AC group. We conclude that SLC-AC is the best protocol to improve sperm quality in chilled canine semen in comparison to the other procedures tested.