This paper presents the results of genetic variability analyses using genealogical data on the main genetic bloodline of the Australian Cattle Dog in Italy, a line that has had a significant impact on the development of the breed. All the genealogical data on the progeny and ancestors of one of the first stallions introduced in Italy were considered, i.e. Cattlefarm's Comeback Jack born on 1/2/1997 in Finland. Animals from the bloodline born between 1962 and 2019 were considered. A total number of 1722 animals were found to be from the line which represents the entire population (WP), including the basic population (BP) and the reference population (RP) defined as the animals currently living. A total of 982 animals were in the RP, with the oldest living dogs born in 2004. A total of 854 dogs were inbred. The average inbreeding coefficient (F) in the RP was 5.1%, while the average inbreeding of the inbred animals was 5.8%. The F was < 0.10 in 711 dogs (77.3% of inbred), and > 0.20 in only 36 dogs (3.91% of inbred). Fifteen traced generations were highlighted. A maximum average inbreeding value (6.45%) was observed in the dogs with 11 traced generations. This research highlighted the good genetic variability of this Australian Cattle Dog bloodline thanks to the efficient management of the breeders who in the past introduced some stallions from abroad. Currently, the lines in Italy are not sufficiently high to prevent inbreeding in the new matings, which is becoming frequent. It is, therefore, important to continue to import new stallions for reproduction to expand the genetic variability. However, at the same time, the old lines need to be preserved genetically, aptitudinally and morphologically, as they are an important heritage of the breed in Italy.