Estrogen receptor (ER) status in breast cancer (BC) is determined using immunohistochemistry (IHC) with nuclear expression in ≥1% of cells defined as ER-positive. BC with 1%-9% expression (ER-low-positive), is a clinically and biologically unique subgroup. In this study, we hypothesized that ER-low-positive BC represents a heterogeneous group with a mixture of ER-positive and ER-negative tumor, which may explain their divergent clinical behavior. A large BC cohort (n = 8171) was investigated and categorized into 3 groups: ER-low-positive (1%-9%), ER-positive (≥10%), and ER-negative (<1%) where clinicopathological and outcome characteristics were compared. A subset of ER-low-positive cases was further evaluated using IHC, RNAscope, and RT-qPCR. PAM50 subtyping and ESR1 mRNA expression levels were assessed in ER-low-positive cases within The Cancer Genome Atlas data set. The reliability of image analysis software in assessment of ER expression in the ER-low-positive category was also assessed. ER-low-positive tumors constituted <2% of BC cases examined and showed significant clinicopathological similarity to ER-negative tumors. Most of these tumors were nonluminal types showing low ESR1 mRNA expression. Further validation of ER status revealed that 45% of these tumors were ER-negative with repeated IHC staining and confirmed by RNAscope and RT-qPCR. ER-low-positive tumors diagnosed on needle core biopsy were enriched with false-positive ER staining. BCs with 10% ER behaved similar to ER-positive, rather than ER-negative or low-positive BCs. Moderate concordance was found in assessment of ER-low-positive tumors, and this was not improved by image analysis. Routinely diagnosed ER-low-positive BC includes a proportion of ER-negative cases. We recommend repeat testing of BC showing 1%-9% ER expression and using a cutoff ≥10% expression to define ER positivity to help better inform treatment decisions.