Deregulation of signal transduction pathways frequently confers selective biological advantages to tumors. Phosphoinositides play an essential role in numerous cellular functions and, among the enzymes implicated in these processes, phosphoinositide-specific phospholipase C β1 (PI-PLCβ1) is one of the key regulators. In the present study, a fluorescence in situ hybridization (FISH) approach was used to investigate PI-PLCβ1 gene copy number alterations in various types of breast cancer differing in their invasiveness and proliferative activity, according to their mitotic index. At the molecular level, we also performed both real-time PCR and immunohistochemical analyses on PI-PLCβ1 to further investigate its expression in primary breast cancers. Finally, we analyzed the correlation between PI-PLCβ1 gene copy number and clinicopathological parameters. Our results show that most of our cases had aneusomies on the PI-PLCβ1 locus (20p12) and amplification of this specific region was the most frequent alteration observed. Our findings also indicate that the amplification of the region containing the PI-PLCβ1 gene was mostly related to the mitotic index, rather than to the invasion status. Finally, even though our case series is limited, PI-PLCβ1 gene amplification seems to be correlated to clinicopathological parameters.