FFPE (formalin-fixed, paraffin-embedded) and Frozen tissue archives are the largest repository of clinically annotated human specimens. Despite numerous advances in technology, current methods for sequencing of FFPE-fixed and Snap-Frozen single-cells are slow, labour intensive, insufficiently sensitive and have a low resolution, making it difficult to fully exploit their enormous research and clinical potential. Here we introduce single nuclei pathology sequencing (snPATHO-seq), a sensitive and efficient high-throughput platform to profile the transcriptome of single nuclei extracted from formalin-fixed paraffin-embedded (FFPE) samples. While some protocols have been made available for the preparation of single cell suspensions from FFPE tissues, the quality of the preparations is variable for reliable single cell analysis. snPATHO-seq combines an optimised nuclei extraction protocol from archival samples with 10x Genomics probe-based technology targeting the whole transcriptome. We applied snPATHO-Seq to study tumour heterogeneity in breast cancer samples, including challenging primary tumours and liver metastases collected at autopsy. On average, snPATHO-Seq on FF samples increased 3.3 times the detection of transcription factors when compared to standard poly A capture, making these data ideal for inference of gene regulatory networks. snPATHO-Seq was also optimized for FFPE samples using a variety of samples, including kidney, prostate, colon, and breast. We generated paired snPATHO-Seq and spatial transcriptomics data from sequential sections to reveal strong gene expression correlations, validating the accuracy of the snPATHO-Seq data. Gene expression data from snPATHO-Seq was used to resolve fine-grained cell types within each spatial transcriptomic location via deconvolution. Overall, snPATHO-Seq enables high quality and sensitivity snRNA-Seq from preserved tissue samples, unlocking the vast pathology archives and thereby allowing extensive retrospective clinical genomic studies.