We evaluated collagen deposition and nuclear phenotypes in non-inflammatory, metastasis-free canine mammary carcinomas at the time of tumour resection. A retrospective cohort analysis was conducted on 68 female dogs diagnosed with mammary carcinomas between January 2013 and December 2021, excluding cases of mammary sarcoma, carcinosarcoma, inflammatory mammary cancer and metastases. Tumours were classified into histological subtypes using the Peña grading system and assigned grades accordingly. Software-assisted video image analysis was utilized to quantitatively assess collagen deposition, organization and nuclear phenotypes. Histological grading was performed by three independent examiners to ensure reproducibility and minimize observer bias. Significant differences in collagen deposition and nuclear phenotypes were observed across histological grades. Grade III carcinomas had significantly greater collagen deposition, both within the tumour core and at the tumour periphery, compared with grades I and II. Collagen organization was markedly increased in grade III carcinomas. Nuclear phenotype analysis revealed distinct features that allowed clear differentiation between grade II and grade III tumours. Software-assisted image analysis effectively identified distinct patterns of collagen deposition, organization and nuclear phenotypes associated with canine mammary carcinomas of various grades, providing important information about tumour biology.